U.S. patent application number 15/532909 was filed with the patent office on 2017-12-14 for tdp-43-binding polypeptides useful for the treatment of neurodegenerative diseases.
The applicant listed for this patent is UNIVERSITE LAVAL. Invention is credited to Claude Gravel, Jean-Pierre Julien, Silvia Pozzi.
Application Number | 20170355756 15/532909 |
Document ID | / |
Family ID | 56090779 |
Filed Date | 2017-12-14 |
United States Patent
Application |
20170355756 |
Kind Code |
A1 |
Julien; Jean-Pierre ; et
al. |
December 14, 2017 |
TDP-43-Binding Polypeptides Useful for the Treatment of
Neurodegenerative Diseases
Abstract
Provided herein are antigen-binding constructs such as
antibodies that bind to the RRM-1 domain of TDP-43. The
antigen-binding constructs are capable of blocking the interaction
of TDP-43 with NF-.kappa.B in cells. Also provided herein are
method of using the antigen-binding constructs in the treatment of
diseases associated with TPD-43 proteinopathy, such as amyotrophic
lateral sclerosis (ALS), frontotemperal lobar degeneration (FTLD),
Lewy body disease and motor neuron disease.
Inventors: |
Julien; Jean-Pierre;
(Quebec, CA) ; Gravel; Claude; (Quebec, CA)
; Pozzi; Silvia; (Quebec, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE LAVAL |
Quebec (Quebec) |
|
CA |
|
|
Family ID: |
56090779 |
Appl. No.: |
15/532909 |
Filed: |
December 7, 2015 |
PCT Filed: |
December 7, 2015 |
PCT NO: |
PCT/CA2015/051280 |
371 Date: |
June 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62088012 |
Dec 5, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/76 20130101;
C12N 15/86 20130101; C07K 2317/82 20130101; C07K 2317/55 20130101;
C07K 2317/52 20130101; C07K 2317/622 20130101; C07K 2317/54
20130101; A61P 25/16 20180101; C12N 2750/14143 20130101; C07K
2317/24 20130101; C07K 2317/569 20130101; A61K 48/00 20130101; C07K
16/18 20130101; A61P 25/28 20180101; A61P 9/10 20180101; A61P 25/00
20180101; A61P 21/02 20180101; C07K 2317/35 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C12N 15/86 20060101 C12N015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 5, 2014 |
CA |
2,874,083 |
Claims
1. An antigen-binding construct that specifically binds a TAR-DNA
binding protein 43 kDa (TDP-43) comprising at least one heavy chain
variable region VH comprising three VH complementarity determining
regions (CDRs), wherein the VH comprises one, two or three of: (a)
a CDR selected from E6_VH1 CDR1, E6_VH7 CDR1, C10_VH3 CDR1 or
C10_VH4 CDR1; (b) a CDR selected from E6_VH1 CDR2, E6_VH7 CDR2,
C10_VH3 CDR2 or C10_VH4 CDR2; and/or (c) a CDR selected from E6_VH1
CDR3, E6_VH7 CDR3, C10_VH3 CDR3 or C10_VH4 CDR3.
2. The antigen-binding construct of claim 1, comprising one, two or
three VH CDRs that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%
or 99% identical to: (a) a CDRH1 selected from E6_VH1 CDR1, E6_VH7
CDR1, C10_VH3 CDR1 and C10_VH4 CDR1; (b) a CDR2H2 selected from
E6_VH1 CDR2, E6_VH7 CDR2, C10_VH3 CDR2 and C10_VH4 CDR2; and/or (c)
a VH CDRH3 selected from E6_VH1 CDR3, E6_VH7 CDR3, C10_VH3 CDR3 and
C10_VH4 CDR3.
3. The antigen-binding construct of claim 1 or 2, comprising the VH
of E6_VH1, E6_VH7, C10_VH3 or C10_VH4.
4. The antigen-binding construct of claim 3, wherein the VH is at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VH
of E6_VH1, E6_VH7, C10_VH3 or C10_VH4.
5. The antigen-binding construct of any one of claims 1-4, further
comprising a variable light chain region VL, wherein the VL
comprises three VL CDRs, and wherein the VL comprises one, two or
three of: (a) a CDR selected from E6_V.kappa.9 CDR1 and
C10_V.kappa.3 CDR1; (b) a CDR selected from E6_V.kappa.9 CDR2 and
C10_V.kappa.3 CDR2; and/or (c) a CDR selected from E6_V.kappa.9
CDR3 or C10_V.kappa.3 CDR3.
6. The antigen-binding construct of claim 5, wherein the VL
comprises one, two or three VL CDRs that are at least 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to: (a) a CDR selected
from E6_V.kappa.9 CDR1 and C10_V.kappa.3 CDR1; (b) a CDR selected
from E6_V.kappa.9 CDR2 and C10_V.kappa.3 CDR2; and/or (c) a CDR
selected from E6_V.kappa.9 CDR3 or C10_V.kappa.3 CDR3.
7. The antigen-binding construct of any one of claims 1-6,
comprising the 6 CDRs selected from: (a) the CDR1 (SEQ ID NO. 7),
CDR2 (SEQ ID NO. 8) and CDR3 (SEQ ID NO. 9), of E6_VH1 and the CDR1
(SEQ ID NO. 7), CDR2 (SEQ ID NO. 8) and CDR3 (SEQ ID NO. 9) of
E_6V.kappa.9; (b) the CDR1 (SEQ ID NO. 10), CDR2 (SEQ ID NO. 11)
and CDR3 (SEQ ID NO12) of E6_VH7 and the CDR1 (SEQ ID NO. 7), CDR2
(SEQ ID NO. 8) and CDR3 (SEQ ID NO. 9) of E_6V.kappa.9; (c) the
CDR1 (SEQ ID NO16), CDR2 (SEQ ID NO. 17) and CDR3 (SEQ ID NO. 18)
of C10_VH3, and the CDR1 (SEQ ID NO. 22), CDR2 (SEQ ID NO. 23 and
CDR3 (SEQ ID NO. 24) of C10_V.kappa.3; or (d) the CDR1 (SEQ ID
NO19), CDR2 (SEQ ID NO. 20) and CDR3 (SEQ ID NO. 21) of C10_VH4 and
the CDR1 (SEQ ID NO. 22), CDR2 (SEQ ID NO. 23 and CDR3 (SEQ ID NO.
24) of C10_V.kappa.3.
8. The antigen-binding construct of any one of claims 5-7 wherein
the antigen-binding polypeptide comprises the VL of E6_V.kappa.9 or
C10_V.kappa.3.
9. The antigen-binding construct of claim 8 wherein the VL is at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VL
of E6_V.kappa.9 or C10_V.kappa.3.
10. The antigen binding construct of any one of claims 5-9,
comprising the VH region of E6_VH1 or E6_VH7 and the VL region of
E6_V.kappa.9.
11. The antigen binding construct of any one of claims 5-9,
comprising the VH region of C10_VH3 or C10_VH4 and the VL region of
C10_V.kappa.3.
12. The antigen-binding construct of any one of claims 5-11,
comprising: i) the VH of E6_VH7 and the VL of E6_V.kappa.9; ii) the
VH of E6_VH1 and the VL of E6_V.kappa.9; iii) the VH of C10_VH3 and
the VL of C10_V.kappa.3; or iv) the VH of C10_VH4 and the VL of
C10_V.kappa.3.
13. The antigen-binding construct of any one of claims 5-12,
wherein the construct comprises a peptide linker between the VH and
VL, optionally wherein the linker comprises the amino acid sequence
SSGGGGSGGGGSGGGGS (SEQ ID NO:47).
14. The antigen-binding construct of any one of claims 5-13,
wherein the construct comprises at least one of E6_Vh7V.kappa.9,
E6_Vh1V.kappa.9, C10_VH3V.kappa.3 and C10_VH4V.kappa.3.
15. The antigen-binding construct of any one of claims 5-13,
wherein the construct comprises E6_Vh7V.kappa.9, E6_Vh1V.kappa.9,
C10_VH3V.kappa.3 or C10_VH4V.kappa.3.
16. The antigen-binding construct of any one of claims 1-15,
wherein the construct comprises a secretory signal peptide.
17. The antigen-binding construct of claim 16, wherein the
secretory signal peptide is M G D N D I H F A F L S T G V H S Q V Q
(SEQ ID NO:48).
18. The antigen-binding construct of any one of claims 5-17,
wherein the construct has an scFv format.
19. The antigen-binding construct of any one of claims 5-18 wherein
the construct has an Fab format.
20. The antigen binding construct of any one of claims 1-4, wherein
the construct has a single domain antibody format.
21. The antigen-binding construct of any one of claims 5-17,
wherein the construct has an (Fab').sub.2 format.
22. The antigen-binding construct of any one of claims 1 to 21,
wherein the construct comprises an Fc domain.
23. The antigen-binding construct of any one of claims 1 to 22,
wherein the construct is humanized.
24. An antigen-binding construct that specifically binds to an
RRM-1 domain of TDP-43, comprising a VH and a VL, wherein the
construct, when expressed in a cell, i) reduces the interaction of
an intracellular TDP-43 polypeptide with an intracellular
NF-.kappa.B p65 polypeptide in Hek293 cells by 10% or more; or ii)
reduces the activation of NF-.kappa.B in the BV-2 cells in response
to LPS by 10% or more; or iii) reduces the level of nuclear TDP-43
in Neuro2A cells; or iv) reduces the lysine acetylation of TDP-43
in Hek293 cells in response to TNF alpha by 10% or more; or v)
reduces the insolubility of TDP-43 in Hek293 cells incubated with
ethacrynic acid.
25. The antigen-binding construct of claim 24, wherein the
construct is any one of claims 5-23.
26. An antigen-binding construct that blocks by 50% or more the
binding of any of E6_VH1Vk9, E6_VH7Vk9, C10_VH3Vk3 or C10_VH4Vk3 to
TDP-43 or to the RRM-1 domain of TDP-43.
27. A pharmaceutical composition comprising the antigen-binding
construct of any one of claims 1 to 25 and a pharmaceutically
acceptable excipient.
28. A method of treating or preventing a disease characterized by
TDP-43 proteinopathy in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
the antigen-binding construct of any one of claims 1-26 or the
pharmaceutical composition of claim 27.
29. A method of treating or preventing a disease characterized by
TDP-43 proteinopathy in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an adeno-associated viral (AAV) vector comprising at least one
nucleic acid sequence that encodes at least one antigen-binding
construct of any one of claims 1-26.
30. Use of the antigen-binding constructs of any one of claims 1-26
for the manufacture of a medicament for the treatment or prevention
of a disease or disorder characterized by TDP-proteinopathy.
31. Use of the antigen-binding constructs of any one of claims 1-26
for the treatment or prevention of a disease or disorder
characterized by TDP-proteinopathy.
32. The method or use of any one of claims 28-31 wherein the
disease or disorder is selected from amyotrophic lateral sclerosis
(ALS), Alzheimer's disease, motor neuron disease, Parkinson's
disease, frontotemperal lobar degeneration (FTLD), mild cognitive
impairment (MCI), Lewy body disease, brain trauma or cerebral
ischemia.
33. The method or use of any one of claims 28-32 wherein the
antigen-binding construct is administered in a pharmaceutical
formulation.
34. A method of producing the antigen-binding constructs of any one
of claims 1-26 comprising culturing a host cell under conditions
suitable for expressing the antigen-binding construct, wherein the
host cell comprises a polynucleotide encoding the antigen binding
construct of any one of claims 1-25, and purifying the
construct.
35. An polynucleotide or set of isolated polynucleotides comprising
at least one nucleic acid sequence that encodes at least one of the
antigen-binding construct of any one of claims 1-26.
36. The polynucleotide of claim 35 wherein the polynucleotide or
set of isolated polynucleotides is cDNA.
37. The polynucleotide or set of isolated polynucleotides of claim
35 or 36, comprising at least one nucleic acid sequence that
encodes: i) the VH of E6_VH7 and/or the VL of E6_V.kappa.9; ii) the
VH of V.kappa.9 and/or the VL of E6_V.kappa.9; iii) the VH of
C10_VH3 and/or the VL of C10_V.kappa.3; or iv) the VH of C10_VH4
and/or the VL of C10_V.kappa.3.
38. A polynucleotide or set of polynucleotides comprising a nucleic
acid sequence that encodes E6_VH7V.kappa.9, E6_VH1V.kappa.9,
C10_VH3V.kappa.3 or C10_VH4V.kappa.3.
39. A vector or set of vectors comprising one or more of the
polynucleotides or sets of polynucleotides of any one of claims
35-38.
40. A vector or set of vectors comprising one of more of the
polynucleotides or sets of polynucleotides of any one of claims
35-38 which is selected from a plasmid, a viral vector, a
non-episomal mammalian vector, an expression vector, and a
recombinant expression vector.
41. The vector of claim 39 or 40 wherein the vector is an
adeno-associated viral (AAV) vector.
42. An isolated cell comprising a polynucleotide or set of
polynucleotides according to any one of claims 35-38, or a vector
or set of vectors of any one of claims 39 to 41.
43. A kit comprising the antigen-binding construct of any one of
claims 1-26 and instructions for use.
Description
[0001] This application relates to TDP43 (TAR DNA-binding protein
of 43 kDA)-specific binding constructs such as antibodies and
fragments thereof that specifically bind to TDP-43. The application
also relates to methods of using the TDP-43-binding polypeptides in
the diagnosis and treatment of diseases characterized by TDP-43
proteinopathy such as amyotrophic lateral sclerosis, Alzheimer's
disease, motor neuron disease, Parkinson's disease and
frontotemporal lobar degeneration.
[0002] Neurodegenerative diseases are characterized by selective
neurodegeneration in specific regions of the brain and spinal cord.
Amyotrophic Lateral Sclerosis (ALS), commonly known as "Lou
Gehrig's disease", is a progressive neurodegenerative disease of
unknown etiology. The disease progressively impairs an individual's
ability to control voluntary muscle movement. The disease tends to
progress rapidly, leading to paralysis and death within 2-5 years
of diagnosis in most cases.
[0003] A relatively recent discovery related to TDP-43 has provided
fundamental insights into pathogenic mechanisms operative in ALS.
TDP-43 was shown to be associated with the p65 sub-unit of the
nuclear factor-.kappa.B (NF-.kappa.B) inflammation-regulating
transcription factor in spinal cord samples obtained from ALS
patients, but not from spinal cord sample of control patients (V.
Swarup et al., 2011, J. Exp. Med., 208:2429-2447).
[0004] There are currently few therapeutic options for patients
suffering from ALS. The only FDA approved drug for the treatment of
ALS is Rilutek.RTM., introduced in 1995, which extends life
expectancy in individuals with ALS for a few months. There is
therefore a need for new therapeutic approaches for
neurodegenerative diseases such as ALS.
[0005] As the symptoms of neurodegenerative diseases characterized
by TDP-43 proteinopathy are similar to those of other neuromuscular
disorders, diseases such as ALS is difficult to diagnose. The
diagnosis is usually based on a complete neurological examination
and clinical tests. There is therefore a need for methods and
reagents for evaluating a subject predisposed to developing a
neurodegenerative disease such as ALS and FTLD-U or suffering from
these neurodegenerative diseases.
[0006] Provided herein are antigen-binding constructs that bind to
TDP-43 (TAR DNA-binding protein of 43 kDa), such as antibodies,
including fragments, derivatives and variants. In one embodiment,
the antigen-binding constructs specifically bind to the RRM-1
domain of TDP-43. In another embodiment, the antigen-binding
constructs, when expressed in a cell, inhibits the binding of
TDP-43 to the p65 subunit of NF-.kappa.B in the cell by at least
5%, 10%, 20%, 30%, 40%, 50% 50%, 70%, 80%, or 90%. In another
embodiment, the antigen-binding constructs, when expressed in
cells, attenuate the activation of NF-.kappa.B in response to LPS
in the cells by at least 5%, 10%, 20%, 30%, 40%, 50% 50%, 70%, 80%,
or 90%. In another embodiment, the antigen-binding constructs, when
expressed in a cell, reduces the level of nuclear TDP-43 in the
cell by at least 5%, 10%, 20%, 30%, 40%, 50% 50%, 70%, 80%, or 90%.
In another embodiment, the antigen-binding constructs, when
expressed in ethacrynic acid-treated Hek293 cells, reduce TDP-43
insolubility by at least 5%, 10%, 20%, 30%, 40%, 50% 50%, 70%, 80%,
or 90%. In another embodiment, the antigen-binding constructs, when
expressed in Hek 293 cells incubated with TNF alpha, reduce the
level of lysine acetylation of cellular TDP-43 by at least 5%, 10%,
20%, 30%, 40%, 50% 50%, 70%, 80%, or 90%.
[0007] Provided herein are antigen-binding constructs that
specifically bind to TDP-43 comprising at least one complementarity
determining region (CDR) selected from the amino acid sequences set
forth in (SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID
NO: 10), (SEQ ID NO: 11), (SEQ ID NO: 12), (SEQ ID NO: 12), (SEQ ID
NO: 14), (SEQ ID NO: 15), (SEQ ID NO: 16), (SEQ ID NO: 17), (SEQ ID
NO: 18), (SEQ ID NO: 19), (SEQ ID NO: 20), (SEQ ID NO: 21), (SEQ ID
NO: 22), (SEQ ID NO: 23) and (SEQ ID NO: 24).
[0008] Also provided herein are antigen-binding constructs that
specifically bind to TDP-43 comprising at least one heavy chain
variable region VH comprising three VH complementarity determining
regions (CDRs), wherein the VH comprises one, two or three of:
[0009] a CDR selected from E6_VH1 CDR1, E6_VH7 CDR1, C10_VH3 CDR1
or C10_VH4 CDR1; [0010] a CDR selected from E6_VH1 CDR2, E6_VH7
CDR2, C10_VH3 CDR2 or C10_VH4 CDR2; and/or [0011] a CDR selected
from E6_VH1 CDR3, E6_VH7 CDR3, C10_VH3 CDR3 or C10_VH4 CDR3.
[0012] In some embodiments, the antigen-binding construct comprises
one, two or three VH CDRs that are at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% identical to these VH CDRs. In some
embodiments, the antigen-binding construct comprises the VH region
of E6_VH1, E6_VH7, C10_VH3 or C10_VH4, or a VH region that is at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VH
of E6_VH1, E6_VH7, C10_VH3 or C10_VH4.
[0013] In some embodiments, the antigen-binding construct further
comprise a variable light chain region VL, wherein the VL comprises
three VL CDRs, and wherein the VL comprises one, two or three of:
[0014] 1. a CDR selected from E6_V.kappa.9 CDR1 and C10_V.kappa.3
CDR1; [0015] 2. a CDR selected from E6_V.kappa.9 CDR2 and
C10_V.kappa.3 CDR2; and/or [0016] 3. a CDR selected from
E6_V.kappa.9 CDR3 or C10_V.kappa.3 CDR3. as well as antigen-binding
constructs comprising a CDR that is at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% identical to these VL CDRs. In some
embodiments, the antigen-binding construct comprises the VL of
E6_V.kappa.9 or C10_V.kappa.3, or an antigen-binding construct
having a VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to the VL of E6_V.kappa.9 or C10_V.kappa.3.
[0017] In some embodiments, the antigen binding construct comprises
the VH region of E6_VH1 or E6_VH7 and the VL region of
E6_V.kappa.9. In other embodiments, the antigen binding construct
comprises the VH region of C10_VH3 or C10_VH4 and the VL region of
C10_V.kappa.3. In other embodiments, the antigen-binding construct
comprises the VH of E6_VH7 and the VL of E6_V.kappa.9, the VH of
E6_VH1 and the VL of E6_V.kappa.9, the VH of C10_VH3 and the VL of
C10_V.kappa.3, or the VH of C10_VH4 and the VL of
C10_V.kappa.3.
[0018] In some embodiments, the antigen-binding construct comprises
a peptide linker between the VH and VL, optionally the amino acid
sequence SSGGGGSGGGGSGGGGS.
[0019] In some embodiments, the antigen-binding construct are
E6_Vh7V.kappa.9, E6_Vh1V.kappa.9, C10_VH3V.kappa.3 or
C10_VH4V.kappa.3.
[0020] In some embodiments, the antigen-binding constructs comprise
the six CDRs of E6_VH7V.kappa.9. In some embodiments, the
antigen-binding constructs comprise the six CDRs of
E6_Vh1V.kappa.9. In some embodiments, the antigen-binding
constructs comprise the six CDRs of C10_VH3V.kappa.3. In some
embodiments, the antigen-binding constructs comprise the six CDRs
of C10_VH4V.kappa.3.
[0021] In some embodiments, the TDP-43 antigen-binding construct
comprises a secretory signal peptide. In a specific embodiment, the
secretory signal peptide is M G D N D I H F A F L S T G V H S Q V
Q. In some embodiments, the TDP-43 antigen-binding construct have
an scFv format. In other embodiments, the TDP-43 antigen-binding
constructs have an Fab format. In one embodiment, the TDP-43
antigen binding construct has a single domain antibody (camelid)
format. In some embodiments, the TDP-43 antigen-binding construct
has an (Fab').sub.2 format. The TDP-43 antigen-binding constructs
may also comprise an Fc domain. The antigen-binding constructs may
also be humanized, or de-immunized.
[0022] Also encompassed herein are antigen-binding constructs that
specifically bind to an RRM-1 domain of TDP-43, comprising a VH and
a VL, such that the construct, when expressed in cells, reduces the
interaction of an intracellular TDP-43 polypeptide with an
intracellular NF-.kappa.B p65 polypeptide, and/or reduces the
activation of NF-.kappa.B in cells in response to LPS. In some
embodiments, the interaction of intracellular TDP-43 polypeptide
with intracellular NF-.kappa.B p65 polypeptide is reduced 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more. In some
embodiments, the activation of NF-.kappa.B in cells in response to
LPS is reduced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%
or more.
[0023] Also provided herein is an antigen-binding construct that
blocks by 50%, 60%, 70%, 80% or 90% or more the binding of any of
E6_VH1Vk9, E6_VH7Vk9, C10_VH3Vk3 or C10_VH4Vk3 to either TDP-43 or
to the RRM-1 domain of TDP-43.
[0024] Also described herein are pharmaceutical compositions
comprising the antigen-binding construct described above, and
pharmaceutically acceptable excipient.
[0025] Also described are methods of treating or preventing a
disease or disorder characterized by TDP-43 proteinopathy in a
subject in need thereof, comprising administering to the subject a
therapeutically effective amount of the TDP-43 antigen-binding
constructs provided herein.
[0026] Provided herein are methods of treating or preventing a
disease or disorder characterized by TDP-43 proteinopathy in a
subject in need thereof, comprising administering to the subject a
therapeutically effective amount of an adeno-associated viral (AAV)
vector comprising at least one nucleic acid sequence that encodes
at least one TDP-43 antigen-binding construct.
[0027] Provided herein is a use of the antigen-binding constructs
as defined herein for the manufacture of a medicament for the
treatment or prevention of a disease or disorder characterized by
TDP-proteinopathy.
[0028] Provided herein is a use of the antigen-binding constructs
as described herein for the treatment or prevention of a disease or
disorder characterized by TDP-proteinopathy.
[0029] In some embodiments, the disease or disorder being treated
is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, motor
neuron disease, Parkinson's disease, frontotemperal lobar
degeneration (FTLD), mild cognitive impairment (MCI), Lewy body
disease, brain trauma or cerebral ischemia.
[0030] Also provided are methods of producing the antigen-binding
constructs comprising culturing a host cell under conditions
suitable for expressing the antigen-binding construct, wherein the
host cell comprises a polynucleotide encoding a TDP-43 antigen
binding construct, and purifying the construct.
[0031] Also provided herein are polynucleotide or set of isolated
polynucleotides comprising at least one nucleic acid sequence that
encodes at least one of the TDP-43 antigen-binding constructs. In
one embodiment, the polynucleotide is a cDNA.
[0032] Also described herein is vector or set of vectors comprising
one or more of the polynucleotides or sets of polynucleotides
encoding the antigen-binding constructs.
[0033] In some embodiments, the vector is a plasmid, a viral
vector, a non-episomal mammalian vector, an expression vector, and
a recombinant expression vector. In a specific embodiment, the
vector is an adeno-associated viral (AAV) vector. Also described is
an isolated host cell comprising one or more polynucleotides
encoding TDP-43 antigen binding constructs.
[0034] FIG. 1 is an immunoblot of nuclear extracts of BV-2 cells
that were fractionated by SDS-PAGE and treated with three exemplary
monocloncal anti-TDP-43 antibodies, C10, G8 and E6, raised against
the RRMI domain of TDP-43. The three antibodies detected TDP-43 in
nuclear extracts of BV-2 cells that were stimulated with LPS or
not.
[0035] FIG. 2 is a bar graph showing the results of an ELISA assay
demonstrating that human recombinant p65_His-Tag directly interacts
with human recombinant TDP-43_GST Tag. The results are shown as the
Absorbance (ABS) at 450 nm read for varying concentrations amount
of human recombinant TDP-43 added to the assay from 0.01 to 0.8
g/ml.
[0036] FIG. 3 is a bar graph showing the results of an ELISA assay
demonstrating that interaction between recombinant TDP-43 and
NF-.kappa.B p65 is blocked by exemplary monoclonal anti-TDP-43
antibodies C10, G8, and E6. The results are shown as the Absorbance
(ABS) at 450 nm read for the different recombinant anti-TDP-43
antibodies.
[0037] FIG. 4 is a schematic representation of vectors encoding
exemplary anti-TDP-43 scFv antibodies. The scFv vectors were
constructed in a VH-linker-V.kappa. format together with a flexible
20-amino acid linker (Gly4Ser)3. The scFv constructs contain a
murine immunoglobulin (Ig) .kappa.-secretory signal for efficient
secretion and a human c-myc epitope to facilitate detection.
[0038] FIG. 5 is an immunoblot of an SDS PAGE gel of nuclear and
cytoplasmic fractions of Hek293 cells transfected with an ScFv9
expression plasmids encoding anti-TDP-43 antigen-binding constructs
E6_VH1V.kappa.9 and E6_VH7V.kappa.9.
[0039] FIG. 6 is a dot blot showing that the E6_VH1V.kappa.9 and
E6_VH7V.kappa.9 antigen-binding constructs bind to amino acids
1-206 of TDP-43.
[0040] FIG. 7 shows the results of an ELISA assay demonstrating
that exemplary anti-TDP-43 antibodies E6_VH1V.kappa.9 and
E6_VH7V.kappa.9 having an scFv format block the interaction between
recombinant TDP-43 and NF-.kappa.B p65.
[0041] FIG. 8 shows that an exemplary anti-TDP-43 antibody
E6_VH1V.kappa.9 in an scFv format co-immunoprecipitate with TDP-43
as detected by polyclonal antibodies against TDP-43. (expressed
into the medium of Hek293 cells following transfection with ScFv9
expression plasmids encoding E6_VH1V.kappa.9).
[0042] FIG. 9 shows that exemplary anti-TDP-43 antibody
E6_VH1V.kappa.9 having an scFv format blocks the interaction of
TDP-43 with NF-.kappa.B p65 in Hek293 cells. This was revealed by
an assay in which cell extracts from Hek293 cells expressing
E6_VH1V.kappa.9 were immunoprecipitated with anti-TDP-43 polyclonal
antibodies, revealing a decreased level of NF-.kappa.B p65 for
cells expressing VH1V.kappa.9 compared to cells transfected with an
empty ScFv9 expression vector.
[0043] FIG. 10 shows the ability of expression vectors encoding
exemplary scFv antibodies against TDP-43 to attenuate NF-.kappa.B
activity in response to LPS as measured by a NF-.kappa.B-luciferase
reporter construct stably integrated into BV2 microglial cells.
[0044] FIG. 11 shows the reduction in levels of nuclear TDP-43
caused by expression of scFv anti-TDP-43 antibodies in Neuro2A
cells.
[0045] FIG. 12 shows the ability of anti-TDP-43 antibody to reduce
TDP-43 insolubility in E6_VH1V.kappa.9 and E6_VH7V.kappa.9
transfected Hek293 cells treated with ethacrynic acid. (A) shows a
western blot. (B) shows a dot blot analysis.
[0046] FIG. 13 shows the ability of anti-TDP-43 antibodies to
protect TDP-43 from lysine acetylation in E6_VH1V.kappa.9 and
E6_VH7V.kappa.9 transfected Hek293 cells.
[0047] Disclosed herein are TDP-43-binding constructs and their
uses in the diagnosis and therapy of neurodegenerative diseases
characterized by TDP-43 proteinopathy.
TDP-43
[0048] As used herein, the terms "TDP43," refers to transactivation
responsive-DNA binding protein of 43 kDa, or TAR-DNA binding
protein of 43kDA, and is used to refer to all types and forms of
TPD-43, including the native form as well as other conformers of
TDP-43, including for example, phosphorylated forms of TDP-43,
aggregates of TDP-43, ubiquitin-associated aggregates of TDP-43 and
TDP-43 variants that have one or more mutations compared to native
TDP-43, and pathogenic forms. TDP-43 also includes fragments of
TDP-43 polypeptide. TDP-43 is a DNA/RNA-binding protein that
contains an N-terminal domain, two RNA-recognition motifs and a
glycine-rich C-terminal domain thought to be important for
mediating protein-protein interactions The amino acid sequence of
human TDP-43 is known in the art; see, e.g., Strausberg et al.,
TARDBP protein (Homo sapiens) GenBank Pubmed: An amino acid
sequence of human TDP 43 is shown in AAH71657 version GL47939520
herein incorporated by reference in its entirety. The amino acid
sequence of native human TDP-43 is as shown below and in Table A
(SEQ ID NO: 29). The RRM1 domain corresponds to amino acids 101 to
176.
TABLE-US-00001 (SEQ ID NO: 49) 1 MSEYIRVTED ENDEPIEIPS EDDGTVLLST
VTAQFPGACG LRYRNPVSQC MRGVRLVEGI 61 LHAPDAGWGN LVYVVNYPKD
NKRKMDETDA SSAVKVKRAV QKTSDLIVLG LPWKTTEQDL 121 KEYFSTFGEV
LMVQVKKDLK TGHSKGFGFV RFTEYETQVK VMSQRHMIDG RWCDCKLPNS 181
KQSQDEPLRS RKVFVGRCTE DMTEDELREF FSQYGDVMDV FIPKPFRAFA FVTFADDQIA
241 QSLCGEDLII KGISVHISNA EPKHNSNRQL ERSGRFGGNP GGFGNQGGFG
NSRGGGAGLG 301 NNQGSNMGGG MNFGAFSINP AMMAAAQAAL QSSWGMMGML
ASQQNQSGPS GNNQNQGNMQ 361 REPNQAFGSG NNSYSGSNSG AAIGWGSASN
AGSGSGFNGG FGSSMDSKSS GWGM
NF-kB p65
[0049] As used herein the term "NFkB p65 or "NF.kappa.B" refers to
nuclear factor kappa B, and "NF-kB p65" or "p65" are used
interchangeably herein to refer to the p65 subunit or chain of NFkB
p65. p.sup.65 polypeptides as well as polynucleotides are known in
the art. For example see NCBI M62399.1. An amino acid sequence for
human NF-.kappa.B p65 is shown below. Reference to p65 herein also
includes fragments of p65.
TABLE-US-00002 (SEQ ID NO: 50)
MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEG
RSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKD
PPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQC
VKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLC
FQVTVRDPSGRPLRLPPVLPHPIFDNRAPNTAELKICRVN
RNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFS
QADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPSDREL
SEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSG
PTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINY
DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMV
SALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEAL
LQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQ
GIPVAPHTTEPMLMEYPEATTRLVTGAQRPPDPAPAPLGA
PGLPNGLLSGDEDFSSIADMDFSALLSQISS
Neurodegenerative Diseases Characterized by TDP-43
Proteinopathy
[0050] The term "TDP-43 proteinopathy" relates to the nervous
system diseases, in particular to neurodegenerative diseases, and
are known as a heterologous group of disorders linked by
association with TDP-43 abnormalities, and particularly with
accumulation and/or aggregation of abnormal or misfolded TDP-43
polypeptides. TDP proteinopathies include, but are not limited to
amyotrophic lateral sclerosis (ALS), Parkinson's disease,
frontotemporal lobar degeneration (FTLD) motor neuron disease,
Alzheimer's disease, dementia with Lewy bodies, Huntington's
disease, or Lewy body disease. Abnormal TDP-43 accumulations may
also be triggered by nerve injury, brain trauma, brain ischemia
(stroke).
[0051] The term "frontotemporal lobar degeneration disease" refers
to a group of disorders associated with atrophy in the frontal and
temporal lobes. Frontotemporal lobar degeneration disease (FTLD)
can include FTLD-tau characterized by tau inclusion, FTLD-TDP43
characterized by ubiquitin and TDP-43 inclusion (FTLD-U), FTLD-FUS
characterized by FUS cytoplasmic inclusions and dementia lacking
distinctive histology (DLDH).
[0052] The term "amyotrophic lateral sclerosis" (ALS) is used
herein to refer to any neurodegenerative disease that usually
attacks both upper and lower motor neurons and causes degeneration
throughout the brain and spinal cord.
Antigen Binding Constructs
[0053] Described herein are antigen-binding constructs that bind to
TDP-43 (TAR DNA-binding protein of 43 kDa), such as antibodies,
including fragments, derivatives and variants of antibodies that
are capable of specifically binding to TDP-43. By "specifically
binding to TDP-43", "antibody specific to/for TDP-43" and
"anti-TDP-43 antibody" and "TDP-43 antibody" is meant specifically,
generally, and collectively, antibodies to TDP-43, or misfolded or
oligomeric or aggregated or posttranslationally modified TDP-43 or
variants of TDP-43. According to one embodiment, antibodies as
described herein (including antigen-binding antibody fragments and
derivatives) specifically binds to the RRM-1 domain of TDP-43. In
one embodiment, the TDP-43 specific antigen binding construct is an
antibody (including antigen-binding fragments or derivatives
thereof) having an immunological binding characteristic of the
antibodies described herein. In some embodiments, the
antigen-binding constructs block the binding of TPD-43 to
NF-.kappa.B p65.
[0054] Described herein are antigen-binding constructs that
specifically bind to TDP-43 comprising at least one heavy chain
variable region VH comprising three VH complementarity determining
regions (CDRs), wherein the VH comprises one, two or three of:
[0055] a CDR selected from E6_VH1 CDR1, E6_VH7 CDR1, C10_VH3 CDR1
or C10_VH4 CDR1; [0056] a CDR selected from E6_VH1 CDR2, E6_VH7
CDR2, C10_VH3 CDR2 or C10_VH4 CDR2; and/or [0057] a CDR selected
from E6_VH1 CDR3, E6_VH7 CDR3, C10_VH3 CDR3 or C10_VH4 CDR3.
[0058] In some embodiments, the antigen-binding construct comprises
one, two or three VH CDRs that are at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% identical to the those VH CDRs. In some
embodiments, the antigen-binding construct comprises the VH of
E6_VH1, E6_VH7, C10_VH3 or C10_VH4, or a VH that is at least 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VH of E6_VH1,
E6_VH7, C10_VH3 or C10_VH4.
[0059] In some embodiments, the antigen-binding construct of
further comprising a variable light chain region VL, wherein the VL
comprises three VL CDRs, and wherein the VL comprises one, two or
three of: [0060] a CDR selected from E6_V.kappa.9 CDR1 and
C10_V.kappa.3 CDR1; [0061] a CDR selected from E6_V.kappa.9 CDR2
and C10_V.kappa.3 CDR2; and/or [0062] a CDR selected from
E6_V.kappa.9 CDR3 or C10_V.kappa.3 CDR3; as well as antigen-binding
constructs comprising a CDR that is at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% identical to those VL CDRs. In some
embodiments, the antigen-binding construct comprises the VL of
E6_V.kappa.9 or C10_V.kappa.3, or an antigen-binding construct
having a VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to the VL of E6_V.kappa.9 or C10_V.kappa.3.
[0063] In some embodiments, the antigen binding construct comprises
the VH region of E6_VH1 or E6_VH7 and the VL region of
E6_V.kappa.9. In other embodiments, the antigen binding construct
comprises the VH region of C10_VH3 or C10_VH4 and the VL region of
C10_V.kappa.3. In other embodiments, the antigen-binding construct
comprises the VH of E6_VH7 and the VL of E6_V.kappa.9, the VH of
E6_VH1 and the VL of E6_V.kappa.9, the VH of C10_VH3 and the VL of
C10_V.kappa.3, or the VH of C10_VH4 and the VL of
C10_V.kappa.3.
[0064] In some embodiments, the antigen-binding construct comprises
the six CDRs selected from:
[0065] (a) the CDR1 (SEQ ID NO. 7), CDR2 (SEQ ID NO. 8) and CDR3
(SEQ ID NO. 9), of E6_VH1 and the CDR1 (SEQ ID NO. 7), CDR2 (SEQ ID
NO. 8) and CDR3 (SEQ ID NO. 9) of E_6V.kappa.9;
[0066] (b) the CDR1 (SEQ ID NO. 10), CDR2 (SEQ ID NO. 11) and CDR3
(SEQ ID NO12) of E6_VH7 and the CDR1 (SEQ ID NO. 7), CDR2 (SEQ ID
NO. 8) and CDR3 (SEQ ID NO. 9) of E_6V.kappa.9;
[0067] (c) the CDR1 (SEQ ID NO16), CDR2 (SEQ ID NO. 17) and CDR3
(SEQ ID NO. 18) of C10_VH3, and the CDR1 (SEQ ID NO. 22), CDR2 (SEQ
ID NO. 23 and CDR3 (SEQ ID NO. 24) of C10_V.kappa.3;
[0068] (d) the CDR1 (SEQ ID NO19), CDR2 (SEQ ID NO. 20) and CDR3
(SEQ ID NO. 21) of C10_VH4 and the CDR1 (SEQ ID NO. 22), CDR2 (SEQ
ID NO. 23 and CDR3 (SEQ ID NO. 24) of C10_V.kappa.3.
[0069] In some embodiments, the antigen-binding construct comprises
a peptide linker between the VH and VL, optionally the amino acid
sequence SSGGGGSGGGGSGGGGS.
[0070] In some embodiments, the antigen-binding construct comprises
E6_Vh7V.kappa.9, E6_Vh1V.kappa.9, C10_VH3V.kappa.3 or
C10_VH4V.kappa.3.
[0071] In some embodiments, the antigen-binding construct comprises
a secretory signal peptide. In a specific embodiment, the secretory
signal peptide is M G D N D I H F A F L S T G V H S Q V Q.
[0072] In some embodiments, the antigen-binding construct have an
scFv format. In other embodiments, the antigen-binding constructs
have an Fab format. In one embodiment, the antigen binding
construct has a single domain antibody (for example, a camelid)
format. In some embodiments, the antigen-binding construct has an
(Fab').sub.2 format. In another embodiment, the antigen-binding
construct has an Fab' format. The antigen-binding constructs may
also comprise an Fc domain. The antigen-binding constructs may also
be humanized, or de-immunized.
[0073] Also provided herein are antigen-binding constructs that
specifically bind to an RRM-1 domain of TDP-43, comprising a VH and
a VL, such that the construct, when expressed in cells, reduces the
interaction of an intracellular TDP-43 polypeptide with an
intracellular NF-.kappa.B p65 polypeptide and/or reduces the
activation of NF-.kappa.B in cells in response to LPS. In some
embodiments, the interaction of intracellular TDP-43 polypeptide
with intracellular NF-.kappa.B p65 polypeptide is reduced 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more. In some
embodiments, the activation of NF-.kappa.B in cells in response to
LPS is reduced by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%
or more.
[0074] Also provided herein are antigen-binding constructs that
specifically bind to an RRM-1 domain of TDP-43, comprising a VH and
a VL, wherein the construct, when expressed in a cell, [0075] i)
reduces the interaction of an intracellular TDP-43 polypeptide with
an intracellular NF-.kappa.B p65 polypeptide in Hek293 cells by 10%
or more; or [0076] ii) reduces the activation of NF-.kappa.B in the
BV-2 cells in response to LPS by 10% or more; or [0077] iii)
reduces the level of nuclear TDP-43 in Neuro2A cells; or [0078] iv)
reduces the lysine acetylation of TDP-43 in Hek293 cells in
response to TNF alpha by 10% or more; or [0079] v) reduces the
insolubility of TDP-43 in Hek293 cells incubated with ethacrynic
acid.
[0080] Also provided herein are antigen-binding construct that
blocks by 50%, 60%, 70%, 80% or 90% or more the binding of any of
E6_VH1Vk9, E6_VH7Vk9, C10_VH3Vk3 or C10_VH4Vk3 to either TDP-43 or
to the RRM-1 domain of TDP-43. In some embodiments, the
antigen-binding construct blocks the binding of any of E6_VH1Vk9,
E6_VH7Vk9, C10_VH3Vk3 or C10_VH4Vk3 to either TDP-43 or to the
RRM-1 domain of TDP-43 by 50% or more.
[0081] The term "antigen binding construct" also refers to any
agent, e.g., polypeptide or polypeptide complex capable of binding
to an antigen. In some aspects an antigen binding construct is a
polypeptide the specifically binds to an antigen of interest. An
antigen binding construct can be a monomer, dimer, multimer, a
protein, a peptide, or a protein or peptide complex; an antibody,
an antibody fragment, or an antigen binding fragment thereof; an
scFv and the like. An antigen binding construct can be a
polypeptide construct that is monospecific, bispecific, or
multispecific. In some aspects, an antigen binding construct can
include, e.g., one or more antigen binding components (e.g., Fabs
or scFvs) linked to one or more Fc. Further examples of antigen
binding constructs are described below and provided in the
Examples.
[0082] The term "bispecific" is intended to include any agent,
e.g., an antigen binding construct or antibody, which has two
different binding specificities.
[0083] The term "multispecific" or "heterospecific" is intended to
include any agent, e.g., an antigen binding construct or antibody,
which has two or more different binding specificities. Accordingly,
embodiments of the antigen-binding constructs described herein, are
inclusive of, but not limited to, bispecific, trispecific,
tetraspecific, and other multispecific molecules.
[0084] An antigen binding construct can be an antibody or antigen
binding portion thereof. As used herein, an "antibody" or
"immunoglobulin" refers to a polypeptide substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, which specifically bind and recognize an analyte (e.g.,
antigen). The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as the myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. The "class" of an
antibody or immunoglobulin refers to the type of constant domain or
constant region possessed by its heavy chain. There are five major
classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of
these may be further divided into subclasses (isotypes), e.g.,
IgGi, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgAi, and IgA.sub.2. The
heavy chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively.
[0085] An exemplary immunoglobulin (antibody) structural unit is
composed of two pairs of polypeptide chains, each pair having one
"light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The
N-terminal domain of each chain defines a variable region of about
100 to 110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these light and heavy chain domains
respectively. The IgG1 heavy chain comprises the VH, CH1, CH2 and
CH3 domains respectively from the N to C-terminus. The light chain
comprises the VL and CL domains from N to C terminus. The IgG1
heavy chain comprises a hinge between the CH1 and CH2 domains. In
certain embodiments, the immunoglobulin constructs comprise at
least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE
connected to a therapeutic polypeptide. In some embodiments, the
immunoglobulin domain found in an antigen binding construct
provided herein, is from or derived from an immunoglobulin based
construct such as a diabody, or a nanobody. In certain embodiments,
the immunoglobulin constructs described herein comprise at least
one immunoglobulin domain from a heavy chain antibody such as a
camelid antibody. In certain embodiments, the immunoglobulin
constructs provided herein comprise at least one immunoglobulin
domain from a mammalian antibody such as a bovine antibody, a human
antibody, a camelid antibody, a mouse antibody or any chimeric
antibody.
[0086] The term "hypervariable region" or "HVR", as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
complementarity determining regions (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. With the exception of CDR1 in VH, CDRs generally
comprise the amino acid residues that form the hypervariable loops.
Hypervariable regions (HVRs) are also referred to as
"complementarity determining regions" (CDRs), and these terms are
used herein interchangeably in reference to portions of the
variable region that form the antigen binding regions. This
particular region has been described by Kabat et al., U.S. Dept. of
Health and Human Services, Sequences of Proteins of Immunological
Interest (1983) and by Chothia et al., J Mol Biol 196:901-917
(1987), where the definitions include overlapping or subsets of
amino acid residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The exact residue numbers which
encompass a particular CDR will vary depending on the sequence and
size of the CDR. Those skilled in the art can routinely determine
which residues comprise a particular CDR given the variable region
amino acid sequence of the antibody.
[0087] An "Fab molecule" or a "Fab" refers to a protein or
polypeptide construct consisting of the VH and CH1 domain of the
heavy chain (the "Fab heavy chain") and the VL and CL domain of the
light chain (the "Fab light chain") of an immunoglobulin. A Fab is
monovalent.
[0088] An "F(ab').sub.2" molecule refers to a protein or
polypeptide construct consisting of two Fabs linked together by
part of a hinge region, but is missing the most of the Fc. An
F(ab').sub.2 molecule may be obtained by digesting an
immunoglobulin with papain or pepsin.
[0089] As used herein, the term "single-chain" refers to a molecule
comprising amino acid monomers linearly linked by peptide bonds. In
certain embodiments, one of the antigen binding moieties is a
single-chain Fab molecule, i.e. a Fab molecule wherein the Fab
light chain and the Fab heavy chain are connected by a peptide
linker to form a single peptide chain. In a particular such
embodiment, the C-terminus of the Fab light chain is connected to
the N-terminus of the Fab heavy chain in the single-chain Fab
molecule. In certain other embodiments, one of the antigen binding
moieties is a single-chain Fv molecule (scFv). As described in more
detail herein, an "scFv" has a variable domain of light chain (VL)
connected from its C-terminus to the N-terminal end of a variable
domain of heavy chain (VH) by a polypeptide chain, or alternately
the C-terminal end of the VH is connected to the N-terminal end of
VL by a polypeptide chain. Antibodies of this type are referred to
herein as having an "scFv format".
[0090] In some embodiments described herein, an scFv format is used
in an antigen-binding construct (i.e. antigen-binding domains
composed of a heavy chain variable domain and a light chain
variable domain). In one embodiment said scFv molecules are human.
In another embodiment said scFv molecules are humanized. In one
embodiment said scFv molecules are murine. The variable regions may
be connected directly or, typically, via a linker peptide that
allows the formation of a functional antigen-binding moiety.
Typical peptide linkers comprise about 2-20 amino acids, and are
described herein or known in the art. Suitable, non-immunogenic
linker peptides include, for example, (G4S)n, (SG4)n, (G4S)n,
G4(SG4)n or G2(SG2)n linker peptides, wherein n is generally a
number between 1 and 10, typically between 2 and 4. The scFv
molecule may be further stabilized by disulfide bridges between the
heavy and light chain variable domains, for example as described in
Reiter et al. (Nat Biotechnol 14, 1239-1245 (1996)). As is known in
the art, scFvs can also be stabilized by mutation of CDR sequences,
as described in [Miller et al., Protein Eng Des Sel. 2010 July;
23(7):549-57; Igawa et al., MAbs. 2011 May-June; 3(3):243-5;
Perchiacca & Tessier, Annu Rev Chem Biomol Eng. 2012;
3:263-86.]. In some embodiments, an TDP-43 antigen-binding
construct in an scFv format is preferred for its ability to cross
cell membranes and enter cells. A schematic representation of an
scFv format antibody is shown in FIG. 4B.
[0091] In some embodiments, a TDP-43 antigen-binding construct may
consist of a single VH polypeptide (camelid format).
[0092] By a "crossover" Fab molecule (also termed "Crossfab") is
meant a Fab molecule wherein either the variable regions or the
constant regions of the Fab heavy and light chain are exchanged,
i.e. the crossover Fab molecule comprises a peptide chain composed
of the light chain variable region and the heavy chain constant
region, and a peptide chain composed of the heavy chain variable
region and the light chain constant region. For clarity, in a
crossover Fab molecule wherein the variable regions of the Fab
light chain and the Fab heavy chain are exchanged, the peptide
chain comprising the heavy chain constant region is referred to
herein as the "heavy chain" of the crossover Fab molecule.
Conversely, in a crossover Fab molecule wherein the constant
regions of the Fab light chain and the Fab heavy chain are
exchanged, the peptide chain comprising the heavy chain variable
region is referred to herein as the "heavy chain" of the crossover
Fab molecule.
[0093] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0094] The antigen-binding constructs may comprise an Fc region or
domain, e.g., a dimeric Fc.
[0095] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an immunoglobulin heavy chain that contains
at least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. Unless otherwise
specified herein, numbering of amino acid residues in the Fc region
or constant region is according to the EU numbering system, also
called the EU index, as described in Kabat et al, Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md., 1991. A "subunit" of
an Fc domain as used herein refers to one of the two polypeptides
forming the dimeric Fc domain, i.e. a polypeptide comprising
C-terminal constant regions of an immunoglobulin heavy chain,
capable of stable self-association. For example, a subunit of an
IgG Fc domain comprises an IgG C.sub.H2 and an IgG C.sub.H3
constant domain.
[0096] In some aspects, the Fc comprises at least one or two
C.sub.H3 sequences. In some aspects, the Fc is a homodimeris Fc. In
some aspects the Fc is heterodimeric FC. In some aspects, the Fc is
a human Fc. In some aspects, the Fc is a murine Fc. In some
aspects, the Fc is a human IgG or IgG1 Fc. In some aspects, the Fc
comprises at least one or two C.sub.H2 sequences.
[0097] In some aspects, the Fc comprises one or more modifications
in at least one of the C.sub.H3 sequences. In some aspects, the Fc
comprises one or more modifications in at least one of the C.sub.H2
sequences. In some aspects, an Fc is a single polypeptide. In some
aspects, an Fc is multiple peptides, e.g., two polypeptides.
[0098] Fused or linked means that the components (e.g. a Fab
molecule or an scFv molecule and an Fc domain subunit) are linked
by peptide bonds, either directly or via one or more peptide
linkers.
[0099] Antigen-binding constructs bind antigen. As used herein, the
term "antigenic determinant" is synonymous with "antigen" and
"epitope," and refers to a site (e.g. a contiguous stretch of amino
acids or a conformational configuration made up of different
regions of non-contiguous amino acids) on a polypeptide
macromolecule to which an antigen-binding moiety binds, forming an
antigen-binding moiety-antigen complex. The antigen bound by the
antigen-binding constructs described herein is TPD-43, and in some
embodiments, the RRM-1 domain of TDP-43.
[0100] "Specifically binds", "specific binding" or "selective
binding" means that the binding is selective for the antigen and
can be discriminated from unwanted or non-specific interactions.
The ability of an antigen-binding moiety to bind to a specific
antigenic determinant can be measured either through an
enzyme-linked immunosorbent assay (ELISA) or other techniques
familiar to one of skill in the art, e.g. surface plasmon resonance
(SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et
al, Glyco J 17, 323-329 (2000)), and traditional binding assays
(Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the
extent of binding of an antigen-binding moiety to an unrelated
protein is less than about 10% of the binding of the
antigen-binding moiety to the antigen as measured, e.g., by SPR. In
certain embodiments, an antigen-binding moiety that binds to the
antigen, or an antigen-binding molecule comprising that
antigen-binding moiety, has a dissociation constant (K.sub.D) of
<1 .mu.M, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01
nM, or <0.001 nM (e.g. 10.sup..about.8 M or less, e.g. from
10.sup..about.8 M to 10''.sup.13 M, e.g., from 10''.sup.9 M to
10''.sup.13 M).
[0101] An antibody "which binds" an antigen of interest is one
capable of binding that antigen with sufficient affinity such that
the antibody is useful as a diagnostic and/or therapeutic
agent.
[0102] "Affinity" refers to the strength of the sum total of
non-covalent interactions between a single binding site of a
molecule (e.g., a receptor) and its binding partner (e.g., a
ligand). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., an
antigen-binding construct and an antigen, or a receptor and its
ligand). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (K.sub.D),
which is the ratio of dissociation and association rate constants
(k.sub.off and k.sub.on, respectively). Thus, equivalent affinities
may comprise different rate constants, as long as the ratio of the
rate constants remains the same. Affinity can be measured by
well-established methods known in the art, including those
described herein. A particular method for measuring affinity is
Surface Plasmon Resonance (SPR).
[0103] The antigen-binding constructs described herein include at
least one antigen-binding polypeptide that binds TDP-43. Table A
shows the amino acid sequences of exemplary anti-TDP-43 antigen
binding polypeptides. ( )=Igk secretory signal; { }=linker
sequence; [ ]=c-myc detection signal.
[0104] Also provided herein are nucleic acid sequences encoding the
exemplary antigen-binding constructs. Table B shows these exemplary
nucleic acid sequences.
TABLE-US-00003 TABLE A SEQUENCES SEQ ID NO DESCRIPTION SEQUENCE
AMINO ACID 1 E6_VH1 heavy chain LQESGGGLVQPGGSMetKLSCVASGFTSSNYWL
variable region NWVRQSPERGLEWVAEIRLKSNNYATNYAESV
KGRFTISRDDSKSSVYLQVNNLRAEDTGIYYCT RSTARATPYYFDYWGQGTTVTV 2 E6_VH7
heavy chain LQQSGGGLVQPGGSMetKLSCVASGFTSSNY variable region
WLNWVRQSPERGLEINVAEIRLKSNNYATNYA ESVKGRFTISRDDSKSSVYLQVNNLRAEDTGI
YYCTRSTARATPYYFDYINGQGTTVTV 3 E6_V.kappa.9 light chain
ELTQSPSSLAVSAGEKVTMetSCKSSQSLLNSRA variable region
RKNFLTWYQQKPGQSPKLLIYWASTRESGVPD RFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYN
LYTFGGGTKLE 4 C10_VH3 heavy chain LQESGGGLVQPGGSRKLSCAASGFTFSSF
variable region GMetHWVRQAPEKGLEINVAYISSGSSTLHYAD
TVKGRFTISRDNPKNTLFLQMetKLPSLCYGLL GPRDHGH 5 C10_VH4 heavy chain
LQQSGGGLVQPGGSRKLSCAASGFTFSSF variable region
GMetHWVRQAPEKGLEINVAYISSGSSTLHYAD TVKGRFTISRDNPKNTLFLQMetKLPSLCYGLL
GPRDHGH 6 C10_V.kappa.3 light chain
ELTQSPASLAVSLGQRATISYRASKSVSTSGY variable region
SYMetHWNQQKPGQPPRLLIYLVSNLESGVPA RFSGSGSGTDFTLNIHPVEEEDAATYYCQHIR
ELTRSEGAPSS 7 E6_VH1 CDR1 SSNYWLNW 8 E6_VH1 CDR2 EIRLKSNNYATNYAE 9
E6_VH1 CDR3 RATPYYFDY 10 E6_VH7 CDR1 SSNYWLNW 11 E6_VH7 CDR2
EIRLKSNNYATNYAE 12 E6_VH7 CDR3 RATPYYFDY 13 E6_V.kappa.9 CDR1
KSSQSLLNSRARKNFLT 14 E6_V.kappa.9 CDR2 YWASTRES 15 E6_V.kappa.9
CDR3 KQSYNLYT 16 C10_VH3 CDR1 SSFGMetHW 17 C10_VH3 CDR2
YISSGSSTLHYAD 18 C10_VH3 CDR3 FLQMetKLPSL 19 C10_VH4 CDR1 SSFGMetHW
20 C10_VH4 CDR2 YISSGSSTLHYAD 21 C10_VH4 CDR3 FLQMetKLPSL 22
C10_V.kappa.3 CDR1 RASKSVSTSGYSYMetH 23 C10_V.kappa.3 CDR2 YLVSNLES
24 C10_V.kappa.3 CDR3 QHIRELTR 25 E6_VH1V.kappa.9 complete
(MGDNDIHFAFLSTGVHSQVQ)LQESGGGLVQPGG with IGk secretory
SMetKLSCVASGFTSSNYWLNWVRQSPERGLE signal and c-myc
WVAEIRLKSNNYATNYAESVKGRFTISRDDSK peptide
SSVYLQVNNLRAEDTGIYYCTRSTARATPYYF
DYWGQGTTVTVSSGG{GGSGGGGSGGGGSDI}ELT
QSPSSLAVSAGEKVTMetSCKSSQSLLNSRARK NFLTWYQQKPGQSPKLLIYWASTRESGVPDR
FTGSGSGTDFTLTISSVQAEDLAVYYCKQSYN LYTFGGGTKLE[EQKLISEEDLN). 26
E6_VH1V.kappa.9 full LQESGGGLVQPGGSMetKLSCVASGFTSSNYW
LNWVRQSPERGLEWVAEIRLKSNNYATNYAE SVKGRFTISRDDSKSSVYLQVNNLRAEDTGIY
YCTRSTARATPYYFDYWGQGTTVTVSSGG{GGS
GGGGSGGGGSDI)ELTQSPSSLAVSAGEKVTMetS CKSSQSLLNSRARKNFLTWYQQKPGQSPKLL
IYWASTRESGVPDRFTGSGSGTDFTLTISSVQ AEDLAVYYCKQSYNLYTFGGGTKLE 27
E6_VH7V.kappa.9 complete (MGDNDIHFAFLSTGVHSQVQ)LQQSGGGLVQPG with
IGk secretory GSMetKLSCVASGFTSSNYWLNWVRQSPERG signal and c-myc
LEWVAEIRLKSNNYATNYAESVKGRFTISRD peptide
DSKSSVYLQVNNLRAEDTGIYYCTRSTARAT PYYFDYWGQGTTVTV{SSGGGGSGGGGSGGGGSD
I}ELTQSPSSLAVSAGEKVTMetSCKSSQSLLN SRARKNFLTWYQQKPGQSPKLLIYWASTRE
SGVPDRFTGSGSGTDFTLTISSVQAEDLAVY YCKQSYNLYTFGGGTKLE[EQKLISEEDLN] 28
E6_VH7V.kappa.9 full LQQSGGGLVQPGGSMetKLSCVASGFTSSNY
WLNWVRQSPERGLEWVAEIRLKSNNYATNY AESVKGRFTISRDDSKSSVYLQVNNLRAEDT
GIYYCTRSTARATPYYFDYWGQGTTVTV{SSG GGGSGGGGSGGGGSDI}ELTQSPSSLAVSAGEKV
TMetSCKSSQSLLNSRARKNFLTWYQQKPGQ SPKLLIYWASTRESGVPDRFTGSGSGTDFTL
TISSVQAEDLAVYYCKQSYNLYTFGGGTKLE 29 C10_VH3V.kappa.3 complete
(MGDNDIHFAFLSTGVHSQVQ)LQESGGGLVQPG with IGk secretory
GSRKLSCAASGFTFSSFGMetHWVRQAPEKG signal and c-myc
LEWVAYISSGSSTLHYADTVKGRFTISRDNP peptide
KNTLFLQMetKLPSLCYGLLGPRDHGH{SSGGG
GSGGGGSGGGGS}ELTQSPASLAVSLGQRATISY RASKSVSTSGYSYMetHWNQQKPGQPPRLLI
YLVSNLESGVPARFSGSGSGTDFTLNIHPVE EEDAATYYCQHIRELTRSEGAPSS[EQKLISEE
DLN] 30 C10_VH3V.kappa.3 full LQESGGGLVQPGGSRKLSCAASGFTFSSF
GMetHWVRQAPEKGLEWVAYISSGSSTLHYA DTVKGRFTISRDNPKNTLFLQMetKLPSLCYG
LLGPRDHGH{SSGGGGSGGGGSGGGGS}ELTQSPA
SLAVSLGQRATISYRASKSVSTSGYSYMetHW NQQKPGQPPRLLIYLVSNLESGVPARFSGSG
SGTDFTLNIHPVEEEDAATYYCQHIRELTRSE GAPSS 31 C10_VH4V.kappa.3 complete
(MGDNDIHFAFLSTGVHSQVQ)LQQSGGGLVCIPG with IGk secretory
GSRKLSCAASGFTFSSFGMetHWVRQAPEKG signal and c-myc
LEWVAYISSGSSTLHYADTVKGRFTISRDNP peptide
KNTLFLQMetKLPSLCYGLLGPRDHGH{SSGGG
GSGGGGSGGGGS}ELTQSPASLAVSLGQRATISY RASKSVSTSGYSYMetHWNQQKPGQPPRLLI
YLVSNLESGVPARFSGSGSGTDFTLNIHPVE EEDAATYYCQHIRELTRSEGAPSS[EQKLISEE
DLN] 32 C10_VH4V.kappa.3 full LQQSGGGLVQPGGSRKLSCAASGFTFSSF
GMetHWVRQAPEKGLEWVAYISSGSSTLHYA DTVKGRFTISRDNPKNTLFLQMetKLPSLCYG
LLGPRDHGH{SSGGGGSGGGGSGGGGS}ELTQSPA
SLAVSLGQRATISYRASKSVSTSGYSYMetHW NQQKPGQPPRLLIYLVSNLESGVPARFSGSG
SGTDFTLNIHPVEEEDAATYYCQHIRELTRSE GAPSS 49 TDP-43 MSEYIRVTED
ENDEPIEIPS EDDGTVLLST VTAQFPGACG LRYRNPVSQC MRGVRLVEGI LHAPDAGWGN
LVYVVNYPKD NKRKMDETDA SSAVKVKRAV QKTSDLIVLG LPWKTTEQDL KEYFSTFGEV
LMVQVKKDLK TGHSKGFGFV RFTEYETQVK VMSQRHMIDG RWCDCKLPNS KQSQDEPLRS
RKVFVGRCTE DMTEDELREF FSQYGDVMDV FIPKPFRAFA FVTFADDQIA QSLCGEDLII
KGISVHISNA EPKHNSNRQL ERSGRFGGNP GGFGNQGGFG NSRGGGAGLG NNQGSNMGGG
MNFGAFSINP AMMAAAQAAL QSSWGMMGML ASQQNQSGPS GNNQNQGNMQ REPNQAFGSG
NNSYSGSNSG AAIGWGSASN AGSGSGFNGG FGSSMDSKSS GWGM 50 NF-.kappa.B p65
MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAG
SIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEA
ELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLC
FQVTVRDPSGRPLRLPPVLPHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDK
VQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPS
DRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVP
SRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPA
PAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDL
GALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEATTRLVTGAQ
RPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS
TABLE-US-00004 TABLE B SEQUENCES SEQ ID NO DESCRIPTION SEQUENCE
NUCLEIC ACID 33 E6_VH1 heavy chain
CTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTC variable region
TCCTGTGTTGCCTCTGGATTCACTTCCAGTAACTACTGGTTGAACTGGGTCCG
CCAGTCTCCAGAGAGGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCT
AATAATTATGCAACAAATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCT
CAAGAGACGATTCCAAAAGTAGTGTCTACCTGCAAGTGAACAACTTAAGAG
CTGAAGACACTGGCATTTATTACTGTACCAGGTCAACAGCTCGGGCTACCCC
ATACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTC 34 E6_VH7 heavy chain
CTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCT variable
region CCTGTGTTGCCTCTGGATTCACTTCCAGTAACTACTGGTTGAACTGGGTCCGC
CAGTCTCCAGAGAGGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTA
ATAATTATGCAACAAATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTC
AAGAGACGATTCCAAAAGTAGTGTCTACCTGCAAGTGAACAACTTAAGAGC
TGAAGACACTGGCATTTATTACTGTACCAGGTCAACAGCTCGGGCTACCCCA
TACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCTC 35 E6_V.kappa.9 light
chain GAGCTCACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCCGGAGAGAAGGTCA variable
region CTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAGCCCGAAAGA
ACTTCTTGACTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAATTGCTGAT
CTATTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGT
GGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACC
TGGCAGTTTATTACTGCAAACAGTCTTATAATCTGTACACGTTCGGAGGGGG CACCAAGCTCGAG
36 C10_VH3 heavy chain
CTGCAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTC variable region
TCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCG
TCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAG
TAGTACCCTCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGA
GACAATCCCAAGAACACCCTGTTCCTGCAAATGAAACTACCCTCACTATGCT
ATGGACTACTGGGGCCAAGGGACCACGGTCACC 37 C10_VH4 heavy chain
CTGCAGCAGTCAGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTC variable region
TCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCG
TCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAG
TAGTACCCTCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGA
GACAATCCCAAGAACACCCTGTTCCTGCAAATGAAACTACCCTCACTATGCT
ATGGACTACTGGGGCCAAGGGACCACGGTCACC 38 C10_V.kappa.3 light chain
GAGCTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCA variable
region CCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATAT
GCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTT
GTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTG
GGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAA
CCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGCACCAAG CTCGAG 39
E6_VH1V.kappa.9 clone
atgggtgacaatgacatccactttgcctttctctccacaggtgtccactcccaggtccaCTGCAGG
complete with Igk
AGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGT secretion
signal and TGCCTCTGGATTCACTTCCAGTAACTACTGGTTGAACTGGGTCCGCCAGTCTC
c-myc peptide CAGAGAGGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTA
TGCAACAAATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGA
CGATTCCAAAAGTAGTGTCTACCTGCAAGTGAACAACTTAAGAGCTGAAGA
CACTGGCATTTATTACTGTACCAGGTCAACAGCTCGGGCTACCCCATACTAC
TTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCtcctcaggtggaggcggttc
aggcggaggtggctctggcggtggcggatcggacatcGAGCTCACCCAGTCTCCATCCT
CCCTGGCTGTGTCAGCCGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCA
GAGTCTGCTCAACAGTAGAGCCCGAAAGAACTTCTTGACTTGGTACCAGCAG
AAACCAGGGCAGTCTCCTAAATTGCTGATCTATTGGGCATCCACTAGGGAAT
CTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCT
CACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAACAG
TCTTATAATCTGTACACGTTCGGAGGGGGCACCAAGCTCGAGatcaaacgggaa
caaaaactcatctcagaagaggatctgaat 40 E6_VH1V.kappa.9 clone full
CTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTC
TCCTGTGTTGCCTCTGGATTCACTTCCAGTAACTACTGGTTGAACTGGGTCCG
CCAGTCTCCAGAGAGGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCT
AATAATTATGCAACAAATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCT
CAAGAGACGATTCCAAAAGTAGTGTCTACCTGCAAGTGAACAACTTAAGAG
CTGAAGACACTGGCATTTATTACTGTACCAGGTCAACAGCTCGGGCTACCCC
ATACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCtcctcaggtgga
ggcggttcaggcggaggtggctctggcggtggcggatcggacatcGAGCTCACCCAGTCT
CCATCCTCCCTGGCTGTGTCAGCCGGAGAGAAGGTCACTATGAGCTGCAAAT
CCAGTCAGAGTCTGCTCAACAGTAGAGCCCGAAAGAACTTCTTGACTTGGTA
CCAGCAGAAACCAGGGCAGTCTCCTAAATTGCTGATCTATTGGGCATCCACT
AGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATT
TCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTG
CAAACAGTCTTATAATCTGTACACGTTCGGAGGGGGCACCAAGCTCGAG 41
E6_VH7V.kappa.9
atgggtgacaatgacatccactttgcctttctctccacaggtgtccactcccaggtccaCTGCAGC
clone complete with Igk
AGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGT secretion
signal and TGCCTCTGGATTCACTTCCAGTAACTACTGGTTGAACTGGGTCCGCCAGTCTC
c-myc peptide CAGAGAGGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTA
TGCAACAAATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGA
CGATTCCAAAAGTAGTGTCTACCTGCAAGTGAACAACTTAAGAGCTGAAGA
CACTGGCATTTATTACTGTACCAGGTCAACAGCTCGGGCTACCCCATACTAC
TTTGACTACTGGGGCCAAGGGACCACGGTCACCTCtcctcaggtggaggcggttca
ggcggaggtggctctggcggtggcggatcggacatcGAGCTCACCCAGTCTCCATCCTC
CCTGGCTGTGTCAGCCGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCA
GAGTCTGCTCAACAGTAGAGCCCGAAAGAACTTCTTGACTTGGTACCAGCAG
AAACCAGGGCAGTCTCCTAAATTGCTGATCTATTGGGCATCCACTAGGGAAT
CTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCT
CACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAACAG
TCTTATAATCTGTACACGTTCGGAGGGGGCACCAAGCTCGAGatcaaacgggaa
caaaaactcatctcagaagaggatctgaat 42 E6_VH7V.kappa.9 clone full
CTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCT
CCTGTGTTGCCTCTGGATTCACTTCCAGTAACTACTGGTTGAACTGGGTCCGC
CAGTCTCCAGAGAGGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTA
ATAATTATGCAACAAATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTC
AAGAGACGATTCCAAAAGTAGTGTCTACCTGCAAGTGAACAACTTAAGAGC
TGAAGACACTGGCATTTATTACTGTACCAGGTCAACAGCTCGGGCTACCCCA
TACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCTCtcctcaggtggagg
cggttcaggcggaggtggctctggcggtggcggatcggacatcGAGCTCACCCAGTCTCC
ATCCTCCCTGGCTGTGTCAGCCGGAGAGAAGGTCACTATGAGCTGCAAATCC
AGTCAGAGTCTGCTCAACAGTAGAGCCCGAAAGAACTTCTTGACTTGGTACC
AGCAGAAACCAGGGCAGTCTCCTAAATTGCTGATCTATTGGGCATCCACTAG
GGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTC
ACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCA
AACAGTCTTATAATCTGTACACGTTCGGAGGGGGCACCAAGCTCGAG 43 C10_VH3V.kappa.3
clone
atgggtgacaatgacatccactttgcctttctctccacaggtgtccactcccaggtccaCTGCAGG
complete with Igk
AGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGC secretion
signal and AGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTC
c-myc peptide CAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCC
TCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCC
CAAGAACACCCTGTTCCTGCAAATGAAACTACCCTCACTATGCTATGGACTA
CTGGGGCCAAGGGACCACGGTCACCtcctcaggtggaggcggttcaggcggaggtggc
tctggcggtggcggatcggacatcGAGCTCACCCAGTCTCCTGCTTCCTTAGCTGTAT
CTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTA
CATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCAC
CCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAG
GTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTG
GAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACA
CGTTCGGAGGGGGCACCAAGCTCGAGatcaaacgggaacaaaaactcatctcagaaga
ggatctgaat 44 C10_VH3V.kappa.3 clone full
CTGCAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTC
TCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCG
TCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAG
TAGTACCCTCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGA
GACAATCCCAAGAACACCCTGTTCCTGCAAATGAAACTACCCTCACTATGCT
ATGGACTACTGGGGCCAAGGGACCACGGTCACCtcctcaggtggaggcggttcagg
cggaggtggctctggcggtggcggatcggacatcGAGCTCACCCAGTCTCCTGCTTCCT
TAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAA
GTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGG
ACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTC
CCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCC
ATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGA
GCTTACACGTTCGGAGGGGGCACCAAGCTCGAG 45 C10_VH4V.kappa.3 clone
atgggtgacaatgacatccactttgcctttctctccacaggtgtccactcccaggtccaCTGCAGC
complete with Igk
AGTCAGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTG secretion
signal and CAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCT
c-myc peptide CCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACC
CTCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATC
CCAAGAACACCCTGTTCCTGCAAATGAAACTACCCTCACTATGCTATGGACT
ACTGGGGCCAAGGGACCACGGTCACCtcctcaggtggaggcggttcaggcggaggtg
gctctggcggtggcggatcggacatcGAGCTCACCCAGTCTCCTGCTTCCTTAGCTGT
ATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAG
TACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCC
ACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGT
GGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTAC
ACGTTCGGAGGGGGCACCAAGCTCGAGatcaaacgggaacaaaaactcatctcagaag
aggatctgaat 46 C10_VH4V.kappa.3 clone full
CTGCAGCAGTCAGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTC
TCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCG
TCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAG
TAGTACCCTCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGA
GACAATCCCAAGAACACCCTGTTCCTGCAAATGAAACTACCCTCACTATGCT
ATGGACTACTGGGGCCAAGGGACCACGGTCACCtcctcaggtggaggcggttcagg
cggaggtggctctggcggtggcggatcggacatcGAGCTCACCCAGTCTCCTGCTTCCT
TAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAA
GTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGG
ACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTC
CCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCC
ATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGA
GCTTACACGTTCGGAGGGGGCACCAAGCTCGAG 47 Linker signal
SSGGGGSGGGGSGGGGS 48 Secretory signal MGDNDIHFAFLSTGVHSQVQ
Polypeptides and Polynucleotides
[0105] The antigen-binding constructs described herein comprise at
least one TDP-43-binding polypeptide. Also described are
polynucleotides encoding the TDP-43-binding polypeptides described
herein.
[0106] As used herein, "isolated" means an agent (e.g., a
polypeptide or polynucleotide) that has been identified and
separated and/or recovered from a component of its natural cell
culture environment. Contaminant components of its natural
environment are materials that would interfere with diagnostic or
therapeutic uses for the antigen-binding construct, and may include
enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. Isolated also refers to an agent that has been
synthetically produced, e.g., via human intervention.
[0107] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. That is, a description directed to a polypeptide applies
equally to a description of a peptide and a description of a
protein, and vice versa. The terms apply to naturally occurring
amino acid polymers as well as amino acid polymers in which one or
more amino acid residues is a non-naturally encoded amino acid. As
used herein, the terms encompass amino acid chains of any length,
including full length proteins, wherein the amino acid residues are
linked by covalent peptide bonds.
[0108] The term "amino acid" refers to naturally occurring and
non-naturally occurring amino acids, as well as amino acid analogs
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally encoded amino acids are
the 20 common amino acids (alanine, arginine, asparagine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, praline,
serine, threonine, tryptophan, tyrosine, and valine) and
pyrrolysine and selenocysteine. Amino acid analogs refers to
compounds that have the same basic chemical structure as a
naturally occurring amino acid, i.e., an a carbon that is bound to
a hydrogen, a carboxyl group, an amino group, and an R group, such
as, homoserine, norleucine, methionine sulfoxide, methionine methyl
sulfonium. Such analogs have modified R groups (such as,
norleucine) or modified peptide backbones, but retain the same
basic chemical structure as a naturally occurring amino acid.
Reference to an amino acid includes, for example, naturally
occurring proteogenic L-amino acids; D-amino acids, chemically
modified amino acids such as amino acid variants and derivatives;
naturally occurring non-proteogenic amino acids such as
.beta.-alanine, ornithine, etc.; and chemically synthesized
compounds having properties known in the art to be characteristic
of amino acids. Examples of non-naturally occurring amino acids
include, but are not limited to, .alpha.-methyl amino acids (e.g.
ca-methyl alanine), D-amino acids, histidine-like amino acids
(e.g., 2-amino-histidine, .beta.-hydroxy-histidine, homohistidine),
amino acids having an extra methylene in the side chain ("homo"
amino acids), and amino acids in which a carboxylic acid functional
group in the side chain is replaced with a sulfonic acid group
(e.g., cysteic acid). The incorporation of non-natural amino acids,
including synthetic non-native amino acids, substituted amino
acids, or one or more D-amino acids into the proteins as described
herein may be advantageous in a number of different ways. D-amino
acid-containing peptides, etc., exhibit increased stability in
vitro or in vivo compared to L-amino acid-containing counterparts.
Thus, the construction of peptides, etc., incorporating D-amino
acids can be particularly useful when greater intracellular
stability is desired or required. More specifically, D-peptides,
etc., are resistant to endogenous peptidases and proteases, thereby
providing improved bioavailability of the molecule, and prolonged
lifetimes in vivo when such properties are desirable. Additionally,
D-peptides, etc., cannot be processed efficiently for major
histocompatibility complex class II-restricted presentation to T
helper cells, and are therefore, less likely to induce humoral
immune responses in the whole organism.
[0109] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0110] Also included are polynucleotides encoding polypeptides of
the antigen-binding constructs. The term "polynucleotide" or
"nucleotide sequence" is intended to indicate a consecutive stretch
of two or more nucleotide molecules. The nucleotide sequence may be
of genomic, cDNA, RNA, semisynthetic or synthetic origin, or any
combination thereof.
[0111] The term "nucleic acid" refers to deoxyribonucleotides,
deoxyribonucleosides, ribonucleosides, or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides which have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless
specifically limited otherwise, the term also refers to
oligonucleotide analogs including PNA (peptidonucleic acid),
analogs of DNA used in antisense technology (phosphorothioates,
phosphoroamidates, and the like). Unless otherwise indicated, a
particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (including but not limited
to, degenerate codon substitutions) and complementary sequences as
well as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes
8:91-98 (1994)).
[0112] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of ordinary skill in the
art will recognize that each codon in a nucleic acid (except AUG,
which is ordinarily the only codon for methionine, and TGG, which
is ordinarily the only codon for tryptophan) can be modified to
yield a functionally identical molecule. Accordingly, each silent
variation of a nucleic acid which encodes a polypeptide is implicit
in each described sequence.
[0113] As to amino acid sequences, one of ordinary skill in the art
will recognize that individual substitutions, deletions or
additions to a nucleic acid, peptide, polypeptide, or protein
sequence which alters, adds or deletes a single amino acid or a
small percentage of amino acids in the encoded sequence is a
"conservatively modified variant" where the alteration results in
the deletion of an amino acid, addition of an amino acid, or
substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar
amino acids are known to those of ordinary skill in the art. Such
conservatively modified variants are in addition to and do not
exclude polymorphic variants, interspecies homologs, and alleles as
described herein.
[0114] Conservative substitution tables providing functionally
similar amino acids are known to those of ordinary skill in the
art. The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and
[0139] 8) Cysteine (C), Methionine (M) (see, e.g., Creighton,
Proteins: Structures and Molecular Properties (W H Freeman &
Co.; 2nd edition (December 1993).
[0115] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same. Sequences are
"substantially identical" if they have a percentage of amino acid
residues or nucleotides that are the same (i.e., about 60%
identity, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, or about 95% identity over a specified region), when
compared and aligned for maximum correspondence over a comparison
window, or designated region as measured using one of the following
sequence comparison algorithms (or other algorithms available to
persons of ordinary skill in the art) or by manual alignment and
visual inspection. This definition also refers to the complement of
a test sequence. The identity can exist over a region that is at
least about 50 amino acids or nucleotides in length, or over a
region that is 75-100 amino acids or nucleotides in length, or,
where not specified, across the entire sequence of a polynucleotide
or polypeptide. A polynucleotide encoding a polypeptide as
described herein, including homologs from species other than human,
may be obtained by a process comprising the steps of screening a
library under stringent hybridization conditions with a labeled
probe having a polynucleotide sequence as described herein or a
fragment thereof, and isolating full-length cDNA and genomic clones
containing said polynucleotide sequence. Such hybridization
techniques are well known to the skilled artisan.
[0116] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0117] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are known to those
of ordinary skill in the art. Optimal alignment of sequences for
comparison can be conducted, including but not limited to, by the
local homology algorithm of Smith and Waterman (1970) Adv. Appl.
Math. 2:482c, by the homology alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity
method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA
85:2444, by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by manual alignment and visual inspection (see, e.g., Ausubel et
al., Current Protocols in Molecular Biology (1995 supplement)).
[0118] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1997)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information available at the World Wide Web at
ncbi.nlm.nih.gov. The BLAST algorithm parameters W, T, and X
determine the sensitivity and speed of the alignment. The BLASTN
program (for nucleotide sequences) uses as defaults a wordlength
(W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of
both strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength of 3, and expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.
Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST
algorithm is typically performed with the "low complexity" filter
turned off.
[0119] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, or less than about
0.01, or less than about 0.001.
[0120] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(including but not limited to, total cellular or library DNA or
RNA).
[0121] The phrase "stringent hybridization conditions" refers to
hybridization of sequences of DNA, RNA, or other nucleic acids, or
combinations thereof under conditions of low ionic strength and
high temperature as is known in the art. Typically, under stringent
conditions a probe will hybridize to its target subsequence in a
complex mixture of nucleic acid (including but not limited to,
total cellular or library DNA or RNA) but does not hybridize to
other sequences in the complex mixture. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Laboratory Techniques in Biochemistry
and Molecular Biology--Hybridization with Nucleic Probes, "Overview
of principles of hybridization and the strategy of nucleic acid
assays" (1993).
[0122] As used herein, the terms "engineer, engineered,
engineering", are considered to include any manipulation of the
peptide backbone or the post-translational modifications of a
naturally occurring or recombinant polypeptide or fragment thereof.
Engineering includes modifications of the amino acid sequence, of
the glycosylation pattern, or of the side chain group of individual
amino acids, as well as combinations of these approaches. The
engineered proteins are expressed and produced by standard
molecular biology techniques.
[0123] By "isolated nucleic acid molecule or polynucleotide" is
intended a nucleic acid molecule, DNA or RNA, which has been
removed from its native environment. For example, a recombinant
polynucleotide encoding a polypeptide contained in a vector is
considered isolated. Further examples of an isolated polynucleotide
include recombinant polynucleotides maintained in heterologous host
cells or purified (partially or substantially) polynucleotides in
solution. An isolated polynucleotide includes a polynucleotide
molecule contained in cells that ordinarily contain the
polynucleotide molecule, but the polynucleotide molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location. Isolated RNA molecules
include in vivo or in vitro RNA transcripts, as well as positive
and negative strand forms, and double-stranded forms. Isolated
polynucleotides or nucleic acids described herein, further include
such molecules produced synthetically, e.g., via PCR or chemical
synthesis. In addition, a polynucleotide or a nucleic acid, in
certain embodiments, include a regulatory element such as a
promoter, ribosome binding site, or a transcription terminator.
[0124] The term "polymerase chain reaction" or "PCR" generally
refers to a method for amplification of a desired nucleotide
sequence in vitro, as described, for example, in U.S. Pat. No.
4,683,195. In general, the PCR method involves repeated cycles of
primer extension synthesis, using oligonucleotide primers capable
of hybridising preferentially to a template nucleic acid.
[0125] By a nucleic acid or polynucleotide having a nucleotide
sequence at least, for example, 95% "identical" to a reference
nucleotide sequence as described herein, it is intended that the
nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence. As a practical matter, whether any particular
polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to a nucleotide sequence as described herein
can be determined conventionally using known computer programs,
such as the ones discussed above for polypeptides (e.g.
ALIGN-2).
[0126] A derivative, or a variant of a polypeptide is said to share
"homology" or be "homologous" with the peptide if the amino acid
sequences of the derivative or variant has at least 50% identity
with a 100 amino acid sequence from the original peptide. In
certain embodiments, the derivative or variant is at least 75% the
same as that of either the peptide or a fragment of the peptide
having the same number of amino acid residues as the derivative. In
certain embodiments, the derivative or variant is at least 85% the
same as that of either the peptide or a fragment of the peptide
having the same number of amino acid residues as the derivative. In
certain embodiments, the amino acid sequence of the derivative is
at least 90% the same as the peptide or a fragment of the peptide
having the same number of amino acid residues as the derivative. In
some embodiments, the amino acid sequence of the derivative is at
least 95% the same as the peptide or a fragment of the peptide
having the same number of amino acid residues as the derivative. In
certain embodiments, the derivative or variant is at least 99% the
same as that of either the peptide or a fragment of the peptide
having the same number of amino acid residues as the
derivative.
[0127] The term "modified," as used herein refers to any changes
made to a given polypeptide, such as changes to the length of the
polypeptide, the amino acid sequence, chemical structure,
co-translational modification, or post-translational modification
of a polypeptide. The form "(modified)" term means that the
polypeptides being discussed are optionally modified, that is, the
polypeptides under discussion can be modified or unmodified.
[0128] In some aspects, an antigen-binding construct comprises an
amino acids sequence that is at least 80, 85, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% identical to a relevant amino acid
sequence or fragment thereof set forth in the Table(s) or accession
number(s) disclosed herein. In some aspects, an isolated
antigen-binding construct comprises an amino acids sequence encoded
by a polynucleotide that is at least 80, 85, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% identical to a relevant nucleotide
sequence or fragment thereof set forth in Table(s) or accession
number(s) disclosed herein.
[0129] In certain embodiments the antigen-binding polypeptide is
derived from humanized, or chimeric versions of these
antibodies.
[0130] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also may comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). In some
embodiments, the anti-TDP-43 antigen binding constructs are
humanized. In one embodiment, the humanized anti-TDP-43 antibodies
comprise the amino acids of murine CDRs.
[0131] De-immunization can also be used to decrease the
immunogemcity of an antibody.
[0132] As used herein, the term "de-immunization" includes
alteration of an antibody to modify T cell epitopes; see, e.g.,
international Application Publication Nos. WO98/52976 and
WO00/34317. For example, VH and VL sequences from the starting
antibody are analyzed and a human T cell epitope "map" from each V
region showing the location of epitopes in relation to
complementarity determining regions (CDRs) and other key residues
within the sequence. Individual T cell epitopes from the T cell
epitope map are analyzed in order to identify alternative amino
acid substitutions with a low risk of altering activity of the
final antibody. A range of alternative VH and VL sequences are
designed comprising combinations of amino acid substitutions and
these sequences are subsequently incorporated into a range of
binding polypeptides, e.g., TDP-43-specific antibodies, including
immunospecific fragments thereof, for use in the diagnostic and
treatment methods disclosed herein, which are then tested for
function. Typically, between 12 and 24 variant antibodies are
generated and tested. Complete heavy and light chain genes
comprising modified V and/or C regions are then cloned into
expression vectors and the subsequent plasmids introduced into cell
lines for the production of whole antibody. The antibodies are then
compared in appropriate biochemical and biological assays, and the
optimal variant is identified.
Pharmaceutical Compositions
[0133] Also provided herein are pharmaceutical compositions
comprising an antigen-binding constructs described herein. Such
compositions comprise the construct and a pharmaceutically
acceptable carrier.
[0134] The term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which the therapeutic is administered. Such pharmaceutical carriers
can be sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. In some
aspects, the carrier is a man-made carrier not found in nature.
Water can be used as a carrier when the pharmaceutical composition
is administered intravenously. Saline solutions and aqueous
dextrose and glycerol solutions can also be employed as liquid
carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like. The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will
contain a therapeutically effective amount of the compound,
preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
patient. The formulation should suit the mode of
administration.
[0135] In certain embodiments, the composition comprising the
construct is formulated in accordance with routine procedures as a
pharmaceutical composition adapted for intravenous administration
to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer.
Where necessary, the composition may also include a solubilizing
agent and a local anesthetic such as lignocaine to ease pain at the
site of the injection. Generally, the ingredients are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0136] In certain embodiments, the compositions described herein
are formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxide
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
Methods of Preparation of TDP-43 Antigen-Binding Constructs
[0137] Also described herein are methods of producing the
anti-TDP-43 antigen-binding constructs. In certain embodiments the
antigen-binding constructs are produced as recombinant molecules by
expression in a cell, e.g., yeast, a microorganism such as a
bacterium, or a human or animal cell line. In embodiments, the
anti-TDP-43 antigen-binding constructs are secreted from the
cells.
[0138] The antigen-binding constructs can be expressed in a host
cell using an expression cassette coding for the antigen-binding
construct. The term "expression cassette" refers to a
polynucleotide generated recombinantly or synthetically, with a
series of specified nucleic acid elements that permit transcription
of a particular nucleic acid in a target cell. The recombinant
expression cassette can be incorporated into a vector, e.g., a
plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or
nucleic acid fragment. Typically, the recombinant expression
cassette portion of an expression vector includes, among other
sequences, a nucleic acid sequence to be transcribed and a
promoter. In certain embodiments, the expression cassette as
described herein comprises polynucleotide sequences that encode
antigen-binding constructs as described herein or fragments
thereof.
[0139] The term "vector" or "expression vector" is synonymous with
"expression construct" and refers to a nucleic acid molecule that
is used to introduce and direct the expression of a specific gene
to which it is operably associated in a target cell. The term
includes the vector as a self-replicating nucleic acid structure as
well as the vector incorporated into the genome of a host cell into
which it has been introduced. The expression vector as described
herein comprises an expression cassette. Expression vectors allow
transcription of large amounts of stable mRNA. Once the expression
vector is inside the target cell, the ribonucleic acid molecule or
protein that is encoded by the gene is produced by the cellular
transcription and/or translation machinery. In one embodiment, the
expression vector as described herein comprises an expression
cassette that comprises polynucleotide sequences that encode
antigen-binding constructs as described herein or fragments
thereof. Exemplary vectors are described herein.
[0140] Typically a host cell is transformed with an expression
vector coding for an antigen-binding construct. "Cell", "host
cell", "cell line" and "cell culture" are used interchangeably
herein and all such terms should be understood to include progeny
resulting from growth or culturing of a cell. "Transformation" and
"transfection" are used interchangeably to refer to the process of
introducing DNA into a cell. Host cells include "transformants" and
"transformed cells," which include the primary transformed cell and
progeny derived therefrom without regard to the number of passages.
In certain embodiments, progeny are not completely identical in
nucleic acid content to a parent cell, but may contain mutations.
Mutant progeny that have the same function or biological activity
as screened or selected for in the originally transformed cell are
included herein.
[0141] A host cell is any type of cellular system that can be used
to generate the antigen-binding constructs as described herein.
Host cells include cultured cells, e.g. mammalian cultured cells,
such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma
cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or
hybridoma cells, bacteria (for example E. coli and Bacillus
subtilis), yeasts (for example Saccharomyces cerevisiae,
Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for
example Aspergillus), insect cells, and plant cells, to name only a
few, but also cells comprised within a transgenic animal,
transgenic plant or cultured plant or animal tissue. Additional
examples of host cells are described herein.
[0142] In some embodiments, the antigen-binding construct is
produced in a mammalian cell. In select embodiments, the mammalian
cell is selected from the group consisting of a VERO, HeLa, HEK,
NSO, Chinese Hamster Ovary (CHO), W138, BHK, COS-7, Caco-2 and MDCK
cell, and subclasses and variants thereof.
Expression Vectors
[0143] Provided are vectors containing polynucleotides encoding an
antigen-binding construct described herein, host cells, and the
production of the antigen-binding construct proteins by synthetic
and recombinant techniques. The vector may be, for example, a
phage, plasmid, viral, or retroviral vector. Retroviral vectors may
be replication competent or replication defective. In the latter
case, viral propagation generally will occur only in complementing
host cells.
[0144] In certain embodiments, the polynucleotides encoding
antigen-binding construct proteins described herein are joined to a
vector containing a selectable marker for propagation in a host.
Generally, a plasmid vector is introduced in a precipitate, such as
a calcium phosphate precipitate, or in a complex with a charged
lipid. If the vector is a virus, it may be packaged in vitro using
an appropriate packaging cell line and then transduced into host
cells.
[0145] In certain embodiments, the polynucleotide insert is
operatively linked to an appropriate promoter, such as the phage
lambda PL promoter, the E. coli lac, trp, phoA and rac promoters,
the SV40 early and late promoters and promoters of retroviral LTRs,
to name a few. Other suitable promoters will be known to the
skilled artisan. The expression constructs will further contain
sites for transcription initiation, termination, and, in the
transcribed region, a ribosome binding site for translation. The
coding portion of the transcripts expressed by the constructs will
preferably include a translation initiating codon at the beginning
and a termination codon (UAA, UGA or UAG) appropriately positioned
at the end of the polypeptide to be translated.
[0146] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418, glutamine synthase, or neomycin resistance for
eukaryotic cell culture, and tetracycline, kanamycin or ampicillin
resistance genes for culturing in E. coli and other bacteria.
Representative examples of appropriate hosts include, but are not
limited to, bacterial cells, such as E. coli, Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast cells
(e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession
No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9
cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma
cells; and plant cells. Appropriate culture mediums and conditions
for the above-described host cells are known in the art.
[0147] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors,
Phagescript vectors, pNH8A, pNH16a, pNH18A; pNH46A, available from
Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and
pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Preferred expression vectors for use in
yeast systems include, but are not limited to pYES2, pYD1,
pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5,
pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from
Invitrogen, Carlsbad, Calif.). Other suitable vectors will be
readily apparent to the skilled artisan.
[0148] In one embodiment, polynucleotides encoding an
antigen-binding construct described herein are fused to signal
sequences that will direct the localization of a protein as
described herein to particular compartments of a prokaryotic or
eukaryotic cell and/or direct the secretion of a protein as
described herein from a prokaryotic or eukaryotic cell. For
example, in E. coli, one may wish to direct the expression of the
protein to the periplasmic space. Examples of signal sequences or
proteins (or fragments thereof) to which the antigen-binding
construct proteins are fused in order to direct the expression of
the polypeptide to the periplasmic space of bacteria include, but
are not limited to, the pelB signal sequence, the maltose binding
protein (MBP) signal sequence, MBP, the ompA signal sequence, the
signal sequence of the periplasmic E. coli heat-labile enterotoxin
B-subunit, and the signal sequence of alkaline phosphatase. Several
vectors are commercially available for the construction of fusion
proteins which will direct the localization of a protein, such as
the pMAL series of vectors (particularly the pMAL-.rho. series)
available from New England Biolabs. In a specific embodiment,
polynucleotides encoding proteins as described herein may be fused
to the pelB pectate lyase signal sequence to increase the
efficiency of expression and purification of such polypeptides in
Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195 and
5,846,818, the contents of which are herein incorporated by
reference in their entireties.
[0149] Examples of signal peptides that are fused to an
antigen-binding construct protein in order to direct its secretion
in mammalian cells include, but are not limited to, the MPIF-1
signal sequence (e.g., amino acids 1-21 of GenBank Accession number
AAB51134), the stanniocalcin signal sequence (MLQNSAVLLLLVISASA
(SEQ ID NO: 51)), and a consensus signal sequence
(MPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 52)). A suitable signal
sequence that may be used in conjunction with baculoviral
expression systems is the gp67 signal sequence (e.g., amino acids
1-19 of GenBank Accession Number AAA72759). In one embodiment, the
signal sequence is an Ig.kappa. secretory signal comprising the
amino acid sequence (M G D N D I H F A F L S T G V H S Q V Q (SEQ
ID NO: 53)). In one embodiment the Ig.kappa. secretory signal is
fused to the N-terminal of an TDP-43-binding polypeptide.
[0150] Vectors which use glutamine synthase (GS) or DHFR as the
selectable markers can be amplified in the presence of the drugs
methionine sulphoximine or methotrexate, respectively. An advantage
of glutamine synthase based vectors are the availability of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine
synthase negative. Glutamine synthase expression systems can also
function in glutamine synthase expressing cells (e.g., Chinese
Hamster Ovary (CHO) cells) by providing additional inhibitor to
prevent the functioning of the endogenous gene. A glutamine
synthase expression system and components thereof are detailed in
PCT publications: WO87/04462; WO86/05807; WO89/10036; WO89/10404;
and WO91/06657, which are hereby incorporated in their entireties
by reference herein. Additionally, glutamine synthase expression
vectors can be obtained from Lonza Biologics, Inc. (Portsmouth,
N.H.). Expression and production of monoclonal antibodies or
antigen-binding constructs using a GS expression system in murine
myeloma cells is described in Bebbington et al., Bio/technology
10:169(1992) and in Biblia and Robinson Biotechnol. Prog.
11:1(1995) which are herein incorporated by reference.
Host Cells
[0151] Also provided are host cells containing vector constructs
described herein, and additionally host cells containing nucleotide
sequences that are operably associated with one or more
heterologous control regions (e.g., promoter and/or enhancer) using
techniques known of in the art. The host cell can be a higher
eukaryotic cell, such as a mammalian cell (e.g., a human derived
cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell can be a prokaryotic cell, such as a bacterial cell. A
host strain may be chosen which modulates the expression of the
inserted gene sequences, or modifies and processes the gene product
in the specific fashion desired. Expression from certain promoters
can be elevated in the presence of certain inducers; thus
expression of the genetically engineered polypeptide may be
controlled. Furthermore, different host cells have characteristics
and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
phosphorylation, cleavage) of proteins. Appropriate cell lines can
be chosen to ensure the desired modifications and processing of the
foreign protein expressed.
[0152] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), human
epithelial kidney 293 cells, and a number of other cell lines. Cell
lines of particular preference are selected through determining
which cell lines have high expression levels and produce antibodies
with constitutive ManLAM binding properties.
[0153] Introduction of the nucleic acids and nucleic acid
constructs as described herein into the host cell can be effected
by calcium phosphate transfection; DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection, or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986). It is
specifically contemplated that the polypeptides as described herein
may in fact be expressed by a host cell lacking a recombinant
vector.
[0154] In addition to encompassing host cells containing the vector
constructs discussed herein, one embodiment also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., the coding
sequence corresponding to a Cargo polypeptide is replaced with an
antigen-binding construct protein corresponding to the Cargo
polypeptide), and/or to include genetic material. The genetic
material operably associated with the endogenous polynucleotide may
activate, alter, and/or amplify endogenous polynucleotides.
[0155] In addition, techniques known in the art may be used to
operably associate heterologous polynucleotides (e.g.,
polynucleotides encoding a protein, or a fragment or variant
thereof) and/or heterologous control regions (e.g., promoter and/or
enhancer) with endogenous polynucleotide sequences encoding a
therapeutic protein via homologous recombination (see, e.g., U.S.
Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication
Number WO 96/29411; International Publication Number WO 94/12650;
Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and
Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each
of which are incorporated by reference in their entireties).
Purification
[0156] Antigen-binding construct proteins described herein can be
recovered and purified from recombinant cell cultures by well-known
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography such as with protein A,
hydroxylapatite chromatography, hydrophobic charge interaction
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography ("HPLC") is employed for
purification.
[0157] In certain embodiments the antigen-binding construct
proteins as described herein are purified using Anion Exchange
Chromatography including, but not limited to, chromatography on
Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toyopearl Q,
Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE,
Fractogel Q and DEAE columns.
[0158] In certain embodiments the proteins described herein may be
purified using Cation Exchange Chromatography including, but not
limited to, SP-sepharose, CM sepharose, poros HS, poros CM,
Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S
and CM columns and their equivalents and comparables.
Chemical Synthesis and Cell-Free Expression
[0159] In addition, antigen-binding construct proteins described
herein can be chemically synthesized using techniques known in the
art (e.g., see Creighton, 1983, Proteins: Structures and Molecular
Principles, W. H. Freeman & Co., N.Y and Hunkapiller et al.,
Nature, 310:105-111 (1984)). For example, a polypeptide
corresponding to a fragment of a polypeptide can be synthesized by
use of a peptide synthesizer. Furthermore, if desired, nonclassical
amino acids or chemical amino acid analogs can be introduced as a
substitution or addition into the polypeptide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4diaminobutyric acid,
alpha-amino isobutyric acid, 4aminobutyric acid, Abu, 2-amino
butyric acid, g-Abu, e-Ahx, 6amino hexanoic acid, Aib, 2-amino
isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, .beta.-alanine, fluoro-amino acids, designer
amino acids such as .beta.-methyl amino acids, C.alpha.-methyl
amino acids, N.alpha.-methyl amino acids, and amino acid analogs in
general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0160] In certain embodiments, cell-free protein expression systems
are utilized to co-express polypeptides (e.g., heavy and light
chain polypeptides) without the use of living cells. Instead, all
components needed to transcribe DNA to RNA and translate the RNA to
protein (e.g. ribosomes, tRNAs, enzymes, cofactors, amino acids)
are provided in solution for use in vitro. In certain embodiments,
the in vitro expression requires (1) the genetic template (mRNA or
DNA) encoding the heavy and light chain polypeptides and (2) a
reaction solution containing the necessary transcriptional and
translational molecular machinery. In certain embodiments, cell
extracts substantially supply components of the reaction solution,
for instance: RNA polymerases for mRNA transcription, ribosomes for
polypeptide translation, tRNA, amino acids, enzymatic cofactors, an
energy source, and cellular components essential for proper protein
folding. Cell-free protein expression systems can be prepared using
lysates derived from bacterial cells, yeast cells, insect cells,
plant cells, mammalian cells, human cells or combinations thereof.
Such cell lysates can provide the correct composition and
proportion of enzymes and building blocks required for translation.
In some embodiments, cell membranes are removed to leave only the
cytosolic and organelle components of the cell.
[0161] Several cell-free protein expression systems are known in
the art as reviewed in Carlson et al. (2012) Biotechnol. Adv.
30:1185-1194. For example, cell-free protein expression systems are
available based on prokaryotic or eukaryotic cells. Examples of
prokaryotic cell-free expression systems include those from E.
coli. Eukaryotic cell-free protein expression systems are available
based on extracts from rabbit reticulocytes, wheat germ, and insect
cells, for example. Such prokaryotic and eukaryotic cell-free
protein expression systems are commercially available from
companies such as Roche, Invitrogen, Qiagen, and Novagen. One
skilled in the art would readily be able to select suitable
cell-free protein expression systems that would produce
polypeptides (e.g., heavy chain and light chain polypeptides) that
are capable of pairing with each other. Further, the cell-free
protein expression system can also be supplemented with chaperones
(e.g. BiP) and isomerases (e.g. disulphide isomerase) to improve
the efficiency of IgG folding.
[0162] In some embodiments, cell-free expression systems are
utilized to co-express the heavy and light chain polypeptides from
DNA templates (transcription and translation) or mRNA templates
(translation only).
Expression in Yeast
[0163] In some embodiments, the antigen-binding construct is
produced in a yeast cell. The yeasts are transformed with a coding
sequence for the desired protein in any of the usual ways, for
example electroporation. Methods for transformation of yeast by
electroporation are disclosed in Becker & Guarente (1990)
Methods Enzymol. 194, 182.
[0164] Useful yeast plasmid vectors include pRS403-406 and
pRS413-416 and are generally available from Stratagene Cloning
Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404,
pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and
incorporate the yeast selectable markers HIS3, 7RP1, LEU2 and URA3.
Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
[0165] Exemplary genera of yeast contemplated to be useful in one
embodiment as hosts for expressing the proteins are Pichua
(formerly classified as Hansenula), Saccharomyces, Kluyveromyces,
Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces,
Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces,
Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia,
Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,
Sporidiobolus, Endomycopsis, and the like. Preferred genera are
those selected from the group consisting of Saccharomyces,
Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora.
Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and
S. rouxii.
[0166] Examples of Kluyveromyces spp. are K. fragilis, K. lactis
and K. marxianus. A suitable Torulaspora species is T. delbrueckii.
Examples of Pichia (Hansenula) spp. are P. angusta (formerly H.
polymorpha), P. anomala (formerly H. anomala) and P. pastoris.
Methods for the transformation of S. cerevisiae are taught
generally in EP 251 744, EP 258 067 and WO 90/01063, all of which
are incorporated herein by reference.
[0167] Exemplary species of Saccharomyces useful for the synthesis
of antigen-binding constructs described herein include S.
cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces
rouxii. Preferred exemplary species of Kluyveromyces include K.
fragilis and K. lactis. Preferred exemplary species of Hansenula
include H. polymorpha (now Pichia angusta), H. anomala (now Pichia
anomala), and Pichia capsulata. Additional preferred exemplary
species of Pichia include P. pastoris. Preferred exemplary species
of Aspergillusinclude A. niger and A. nidulans. Preferred exemplary
species of Yarrowia include Y. lipolytica. Many preferred yeast
species are available from the ATCC. For example, the following
preferred yeast species are available from the ATCC and are useful
in the expression of proteins: Saccharomyces cerevisiae, Hansen,
teleomorph strain BY4743 yap3 mutant (ATCC Accession No. 4022731);
Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 hsp150
mutant (ATCC Accession No. 4021266); Saccharomyces cerevisiae
Hansen, teleomorph strain BY4743 pmt1 mutant (ATCC Accession No.
4023792); Saccharomyces cerevisiae Hansen, teleomorph (ATCC
Accession Nos. 20626; 44773; 44774; and 62995); Saccharomyces
diastaticus Andrews et Gilliland ex van der Walt, teleomorph (ATCC
Accession No. 62987); Kluyveromyces lactis (Dombrowski) van der
Walt, teleomorph (ATCC Accession No. 76492); Pichia angusta
(Teunisson et al.) Kurtzman, teleomorph deposited as Hansenula
polymorpha de Morais et Maia, teleomorph (ATCC Accession No.
26012); Aspergillus niger van Tieghem, anamorph (ATCC Accession No.
9029); Aspergillus niger van Tieghem, anamorph (ATCC Accession No.
16404); Aspergillus nidulans (Eidam) Winter, anamorph (ATCC
Accession No. 48756); and Yarrowia lipolytica (Wickerham et al.)
van der Walt et von Arx, teleomorph (ATCC Accession No.
201847).
[0168] Suitable promoters for S. cerevisiae include those
associated with the PGKI gene, GAL1 or GAL10 genes, CYCI, PH05,
TRP1, ADH1, ADH2, the genes for glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, triose phosphate isomerase, phosphoglucose
isomerase, glucokinase, alpha-mating factor pheromone, [a mating
factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDI
promoter, and hybrid promoters involving hybrids of parts of 5'
regulatory regions with parts of 5' regulatory regions of other
promoters or with upstream activation sites (e.g. the promoter of
EP-A-258 067).
[0169] Convenient regulatable promoters for use in
Schizosaccharomyces pombe are the thiamine-repressible promoter
from the nmt gene as described by Maundrell (1990) J. Biol. Chem.
265, 10857-10864 and the glucose repressible jbp1 gene promoter as
described by Hoffman & Winston (1990) Genetics 124,
807-816.
[0170] Methods of transforming Pichia for expression of foreign
genes are taught in, for example, Cregg et al. (1993), and various
Phillips patents (e.g. U.S. Pat. No. 4,857,467, incorporated herein
by reference), and Pichia expression kits are commercially
available from Invitrogen BV, Leek, Netherlands, and Invitrogen
Corp., San Diego, Calif. Suitable promoters include AOX1 and AOX2.
Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465 include
information on Hansenula vectors and transformation, suitable
promoters being MOX1 and FMD1; whilst EP 361 991, Fleer et al.
(1991) and other publications from Rhone-Poulenc Rorer teach how to
express foreign proteins in Kluyveromyces spp., a suitable promoter
being PGKI.
[0171] The transcription termination signal is preferably the 3'
flanking sequence of a eukaryotic gene which contains proper
signals for transcription termination and polyadenylation. Suitable
3' flanking sequences may, for example, be those of the gene
naturally linked to the expression control sequence used, i.e. may
correspond to the promoter. Alternatively, they may be different in
which case the termination signal of the S. cerevisiae ADHI gene is
preferred.
[0172] In certain embodiments, the desired antigen-binding
construct protein is initially expressed with a secretion leader
sequence, which may be any leader effective in the yeast chosen.
Leaders useful in S. cerevisiae include that from the mating factor
alpha polypeptide (MF.alpha.-1) and the hybrid leaders of EP-A-387
319. Such leaders (or signals) are cleaved by the yeast before the
mature protein is released into the surrounding medium. Further
such leaders include those of S. cerevisiae invertase (SUC2)
disclosed in JP 62-096086 (granted as 911036516), acid phosphatase
(PH05), the pre-sequence of MF.alpha.-1, 0 glucanase (BGL2) and
killer toxin; S. diastaticus glucoarnylase Il; S. carlsbergensis
.alpha.-galactosidase (MEL1); K. lactis killer toxin; and Candida
glucoarnylase.
Post-Translational Modifications
[0173] In certain embodiments are antigen-binding constructs
described herein, which are differentially modified during or after
translation. In some embodiments, the modification is at least one
of: glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage and linkage to an antibody molecule or antigen-binding
construct or other cellular ligand. In some embodiments, the
antigen-binding construct is chemically modified by known
techniques, including but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; and metabolic synthesis in the presence of
tunicamycin.
[0174] Additional post-translational modifications of
antigen-binding constructs described herein include, for example,
N-linked or O-linked carbohydrate chains, processing of N-terminal
or C-terminal ends), attachment of chemical moieties to the amino
acid backbone, chemical modifications of N-linked or O-linked
carbohydrate chains, and addition or deletion of an N-terminal
methionine residue as a result of procaryotic host cell expression.
The antigen-binding constructs described herein are modified with a
detectable label, such as an enzymatic, fluorescent, isotopic or
affinity label to allow for detection and isolation of the protein.
In certain embodiments, examples of suitable enzyme labels include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include iodine, carbon, sulfur, tritium, indium,
technetium, thallium, gallium, palladium, molybdenum, xenon,
fluorine.
[0175] In specific embodiments, antigen-binding constructs
described herein are attached to macrocyclic chelators that
associate with radiometal ions.
[0176] In some embodiments, the antigen-binding constructs
described herein are modified by either natural processes, such as
post-translational processing, or by chemical modification
techniques which are well known in the art. In certain embodiments,
the same type of modification may be present in the same or varying
degrees at several sites in a given polypeptide. In certain
embodiments, polypeptides from antigen-binding constructs described
herein are branched, for example, as a result of ubiquitination,
and in some embodiments are cyclic, with or without branching.
Cyclic, branched, and branched cyclic polypeptides are a result
from posttranslation natural processes or made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990);
Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[0177] In certain embodiments, antigen-binding constructs described
herein are attached to solid supports, which are particularly
useful for immunoassays or purification of polypeptides that are
bound by, that bind to, or associate with proteins as described
herein. Such solid supports include, but are not limited to, glass,
cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride
or polypropylene.
Use of the Antigen-Binding Constructs in the Treatment of
Neurodegenerative Diseases
[0178] In certain embodiments, provided is a method of treating a
disease or disorder characterized by TDP-43 proteinopathy
comprising administering to a subject in which such treatment,
prevention or amelioration is desired, an TDP-43 antigen-binding
construct described herein, in an amount effective to treat,
prevent or ameliorate the disease or disorder.
[0179] "Disorder" or "disease" refers to any condition that would
benefit from treatment with an anti-TDP-43 antibody or method as
described herein. This includes chronic and acute disorders or
diseases including those pathological conditions which predispose
the mammal to the disorder in question. In a preferred embodiment,
the diseases being treated with the antigen-binding constructs
described herein are associated with TDP proteinopathy. These
include, without limitation, amyotophic lateral sclerosis (ALS),
Parkinson's disease, frontotemporal lobar degeneration (FTLD) motor
neuron disease, Alzheimer's disease, dementia with Lewy bodies,
Huntington's disease, Lewy body disease, mild cognitive impairment
(MCI), or TDP-43 abnormalities triggered by nerve injury, brain
trauma, brain ischemia (stroke).
[0180] The term "subject" refers to an animal, in some embodiments
a mammal, which is the object of treatment, observation or
experiment. An animal may be a human, a non-human primate, a
companion animal (e.g., dogs, cats, and the like), farm animal
(e.g., cows, sheep, pigs, horses, and the like) or a laboratory
animal (e.g., rats, mice, guinea pigs, and the like).
[0181] The term "mammal" as used herein includes but is not limited
to humans, non-human primates, canines, felines, murines, bovines,
equines, and porcines. In some embodiments, the subject being
treated with the anti-TDP-43 antigen-binding constructs is a mouse
or a human.
[0182] "Treatment" refers to clinical intervention in an attempt to
alter the natural course of the individual or cell being treated,
and can be performed either for prophylaxis or during the course of
clinical pathology. Desirable effects of treatment include
preventing occurrence or recurrence of disease, alleviation of
symptoms, diminishing of any direct or indirect pathological
consequences of the disease, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments,
antigen-binding constructs as described herein are used to delay
development of a disease or disorder.
[0183] Desirable effects of treatment include, but are not limited
to, preventing occurrence or recurrence of disease, alleviation of
symptoms, diminishment of any direct or indirect pathological
consequences of the disease, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, constructs
described herein are used to delay development of a disease or to
slow the progression of a disease.
[0184] The term "effective amount" as used herein refers to that
amount of construct being administered, which will accomplish the
goal of the recited method, e.g., relieve to some extent one or
more of the symptoms of the disease, condition or disorder being
treated. The amount of the composition described herein which will
be effective in the treatment, inhibition and prevention of a
disease or disorder associated with aberrant expression and/or
activity of a therapeutic protein can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances.
[0185] Effective doses are extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0186] The TDP-43 antigen-binding constructs described herein are
administered to the subject. Various delivery systems are known and
can be used to administer an antigen-binding construct formulation
described herein, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the
compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.
Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid
as part of a retroviral or other vector, etc. Methods of
introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intrathecal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
or compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local.
[0187] In addition, in certain embodiments, it is desirable to
introduce the antigen-binding construct compositions described
herein into the central nervous system by any suitable route,
including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. Pulmonary administration can also be
employed, e.g., by use of an inhaler or nebulizer, and formulation
with an aerosolizing agent.
[0188] In a specific embodiment, it is desirable to administer the
antigen-binding constructs, or compositions described herein
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antigen-binding construct, as
described herein, care must be taken to use materials to which the
protein does not absorb.
[0189] In another embodiment, the antigen-binding constructs or
composition can be delivered in a vesicle, in particular a liposome
(see Langer, Science 249:1527-1533 (1990); Treat et al., in
Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365
(1989); Lopez-Berestein, ibid., pp. 317-327; see generally
ibid).
[0190] In yet another embodiment, the antigen-binding constructs or
composition can be delivered in a controlled release system. In one
embodiment, a pump may be used (see Langer, supra; Sefton, CRC
Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190
(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al.,
J. Neurosurg. 71:105 (1989)). In yet another embodiment, a
controlled release system can be placed in proximity of the
therapeutic target, e.g., the brain, thus requiring only a fraction
of the systemic dose (see, e.g., Goodson, in Medical Applications
of Controlled Release, vol. 2, pp. 115-138 (1984)).
Gene Therapy
[0191] In some embodiments, nucleic acid encoding antigen-binding
constructs described herein can be administered in vivo to promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic acid expression vector and administering it so
that it becomes intracellular, e.g., by use of a retroviral vector
(see U.S. Pat. No. 4,980,286), or by direct injection, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, or by administering it in linkage to a homeobox-like
peptide which is known to enter the nucleus (see e.g., Joliot et
al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc.
Alternatively, a nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression, by homologous
recombination.
[0192] In a specific embodiment, the nucleic acid encoding the
antigen-binding construct is inserted into an adeno-associated
virus (AAV) vector (see Patel et al., Molecular Therapy 22 (3)
498-510 (2014) and Example 12 herein). The viral vector may be
administered to the subject systemically or locally, for example by
intrathecal injection.
[0193] In certain embodiments an antigen-binding construct
described herein is administered as a combination with
antigen-binding constructs with non-overlapping binding target
epitopes on TDP-43.
[0194] The amount of the antigen-binding construct which will be
effective in the treatment, inhibition and prevention of a disease
or disorder can be determined by standard clinical techniques. In
addition, in vitro assays may optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed in
the formulation will also depend on the route of administration,
and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. Effective doses are extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0195] Also included herein is a method of preventing or delaying
the onset of at least one symptom of a TDP-43 proteinopathy in a
subject in need thereof comprising administering a therapeutically
effective amount of an TDP-43 antigen binding construct described
herein. In one embodiment, the subject is an experimental organism,
such as, but not limited to, a transgenic mouse. In one embodiment,
the subject is a human.
[0196] The antigen-binding constructs described herein may be
administered alone or in combination with other types of treatments
used for neurodegenerative diseases, such as Rilutek.RTM..
Methods of Characterizing Antigen-Binding Constructs
[0197] TDP-43 antigen-binding constructs described herein can be
characterized using any in vivo or in vitro models of TDP-43
proteinopathies. A person skilled in the art understands that an
TDP-43 antigen-binding construct can be characterized in a mouse
model for TDP-43 proteinopathies, for example, one of the animal
models for TDP-43 proteinopathies described in Swarup et al. Brain
134: 2610-2626 (2011). Swarup et al. 2011 describes three
transgenic mouse models for TDP proteinopathies: wild type, G348C
and A315T. These transgenic mice exhibit motor and cognitive
impairment (as measured by the Barnes maze test, the step through
passive avoidance test and the accelerating rotarod test).
Additionally, the mice exhibit cytoplasmic TDP-43-positive
ubiquitinated inclusions, intermediate filament abnormalities,
axonopathy and neuroinflammation. Additional animal models are
described in Wegorzewska et al, Proc. Natl. Acad. Sci. U.S.A. 106
(2009), 18809-14; Gurney et al, Science 264 (1994), 1 772-75; Shan
et al, Neuropharmacol. Letters 458 (2009), 70-74; Wils et al, Proc.
Natl. Acad. Sci. USA. 106 (2010), 3858-63; Duchen and Strich, J.
Neurol. Neurosurg, Psychiatry 31 (1968), 535-42; Dennis and Citron,
Neuroscience 185 (2009), 745-50; Swamp et al, Brain 134 (2011),
2610-2626; Sgaz et al, J Clin invest. 121(2):726-38 (2011); Caccamo
et al. Am J Pathol. 180(1):293-302 (2012), Cannon et al, Acta
Neuropathol. 123(6):807-23 (2012), Custer et al, Hum Moi Genet.
19(9): 1741-55 (2010): and Tatom et al, oL Ther. 17 (2009),
607-613. These animal models may be used to evaluate the TDP-43
antigen-binding constructs described herein.
[0198] An experimental model of TDP-43 proteinopathy can be used in
a preventative setting or it can be used in a therapeutic setting.
In a preventative setting, the dosing of animals starts prior to
the onset of the TDP-43 proteinopathy or symptoms thereof. An
TDP-43 antigen-binding construct described herein may be evaluated
for its ability to prevent, reduce or delay the onset of TDP-43
proteinopathy or symptoms thereof. In a therapeutic model, the
dosing of animals starts after the onset of TDP-43 proteinopathy or
a symptom thereof. In a therapeutic setting, an TDP-43
antigen-binding constructs is evaluated for its ability to treat,
reduce or alleviate the TDP-43 proteinopathy or a symptom thereof.
Symptoms of the TDP-43 proteinopathies include, but are not limited
to, accumulation of pathological TDP-43 deposits, pathological
TDP-43 distribution, phosphorylated TDP-43, or insoluble TDP-43
fractions in the neurons, brain, spinal cord, cerebrospinal fluid
or serum of the experimental object. A positive preventative or
therapeutic outcome in any animal model of TDP-43 proteinopathies
indicates that the particular TDP-43 antigen-binding construct can
be used for preventative or therapeutic purposes in a subject other
than the experimental model organism, for example, it can be used
to treat TDP-43 proteinopathies in a human subject in need
thereof.
[0199] In one embodiment, an TDP-43 antigen-binding construct can
be administered to a TDP-43 proteinopathy mouse model and
corresponding control wild type mice. The antigen-binding construct
administered can be a murine antibody, or a human-murine chimera.
The TDP-43 antigen-binding constructs can be administered by any
means known in the art, for example, by intraperitoneal,
intracranial, intramuscular, intrathecal, intravenous,
subcutaneous, oral, and aerosol administration. Experimental
animals can be given one, two, three, four, five or more doses of
the TDP-43 antigen-binding constructs or a control composition,
such as PBS. In one embodiment, experimental animals will be
administered one or two doses of an TDP-43 antigen-binding
construct. In another embodiment, the animals are chronically dosed
with the TDP-43 antigen-binding constructs over several weeks or
months. A skilled, artisan can readily design a dosing regimen that
fits the experimental purpose, for example, dosing regimen for
acute studies, dosing regimen for chronic studies, dosing regimen
for toxicity studies, dosing regimen for preventative or
therapeutic studies. The presence of the TDP-43 antigen-binding
constructs in a particular tissue compartment of the experimental
animals, for example, but not limited to, serum, blood,
cerebrospinal fluid, brain or spinal cord tissue, can be
established using well know methods of the art. In one embodiment,
a TDP-43 antigen-binding construct is capable of penetrating the
blood brain barrier. In another embodiment, a TDP-43
antigen-binding constructs is capable of entering neurons. By
adjusting the dose of the TDP-43 antigen-binding construct and the
dosing frequency, a desired concentration can be maintained in the
experimental animals. Any effect of a TDP-43 antigen-binding
construct as described herein in the TDP-43 proteinopathy models
can be assessed by comparing the level, biochemical characteristics
or distribution of TDP-43 in the treated and control animals. In
one embodiment, a TDP-43 antigen-binding construct is capable of
reducing the level, amount or concentration of TDP-43 inclusions in
the brain or spinal cord in an animal model. The construct can
reduce the level, amount or concentration of TDP-43 inclusions by
at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In another
embodiment, a TDP-43 antigen-binding construct is capable of
reducing the number or frequency of TDP-43 inclusion-positive
neurons in the brain or spinal cord in an animal model, for
example, by at least about 5%, 10%, 20%, 30%, 50% o, 70%, 90% or
more. The effect of a TDP-43 antigen-binding construct can also be
assessed by examining the distribution and biochemical properties
of TDP-43 following administration. In one embodiment, a TDP-43
antigen-binding construct is capable of reducing the amount or
concentration of cytoplasmic or nuclear TDP-43 protein in the brain
or spinal cord of an animal model, for example, by at least about
5%, 10%, 20%, 30%, 50%, 70%, 90% or more, in another embodiment, it
is capable of reducing the amount or concentration of neuritic
TDP-43 protein in the brain or spinal cord of an animal model, for
example, by at least about 5%, 10%, 20%, 30%, 50%, 70%. 90% or
more, in a further embodiment, it can reduce the amount or
concentration of phosphorylated TDP-43 protein in the brain or
spinal cord in an animal model, for example, by at least about 5%,
10%, 20%, 30%, 50%, 70%, 90% or more. Phosphorylated TDP-43 can be
detected using antibodies specific for pathologically
phosphorylated forms of TDP-43, such as p403/p404 and p409/p410.
Hasegawa et al., Ann Neurol, 64: 60-70 (2008). A TDP-43
antigen-binding construct can also alter, for example, reduce or
increase TDP-43 concentration in the blood, serum or cerebrospinal
fluid of an animal model, for example, by at least about 5%, 10%,
20%, 30%, 50%, 70%, 90% or more, in one embodiment, the % reduction
or increase is relative compared to the level, number, frequency,
amount or concentration that existed before treatment, or to the
level, number, frequency, amount or concentration that, exist in an
untreated/control treated subject.
[0200] In one embodiment, a TDP-43 antigen-binding construct can
prevent or delay the onset of at least one symptom of a TDP-43
proteinopathy in a subject. In one embodiment, a TDP-43
antigen-binding construct can reduce or eliminate at least one
symptom of a TDP-43 proteinopathy in a subject. The symptom can be
the formation of pathological TDP-43 deposits, phosphorylated
TDP-43 deposits, or insoluble TDP-43 deposits. The symptom can also
be the presence, or elevated concentration or amount, of TDP-43 in
the serum, blood, urine or cerebrospinal fluid, wherein elevated
concentration amount is compared to a healthy subject. In one
specific embodiment, the symptom is the presence of
TDP-43-associated NF-kB. The symptom can be a neurological
symptom., for example, loss of motor function or cognitive
impairment. In one embodiment, memory impairment is assessed using
the Barnes maze test or the step through passive avoidance test. In
one embodiment motor function impairment is assessed using the
accelerating rotarod test. In one embodiment, at least one symptom
is reduced by at least about 5%, 10%, 15%, 20%, 30%, 50%, 70%, or
90%. In another embodiment, the latency time on the rotarod
apparatus is significantly higher in a treated subject than in a
control subject. In a specific embodiment, the rotarod latency time
is increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90%.
Kits and Articles of Manufacture
[0201] Also described herein are kits comprising one or more
anti-TDP-43 antigen binding constructs. Individual components of
the kit would be packaged in separate containers and, associated
with such containers, can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale. The kit may
optionally contain instructions or directions outlining the method
of use or administration regimen for the antigen binding
construct.
[0202] When one or more components of the kit are provided as
solutions, for example an aqueous solution, or a sterile aqueous
solution, the container means may itself be an inhalant, syringe,
pipette, eye dropper, or other such like apparatus, from which the
solution may be administered to a subject or applied to and mixed
with the other components of the kit.
[0203] The components of the kit may also be provided in dried or
lyophilized form and the kit can additionally contain a suitable
solvent for reconstitution of the lyophilized components.
Irrespective of the number or type of containers, the kits
described herein also may comprise an instrument for assisting with
the administration of the composition to a patient. Such an
instrument may be an inhalant, nasal spray device, syringe,
pipette, forceps, measured spoon, eye dropper or similar medically
approved delivery vehicle.
[0204] In another aspect described herein, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a T cell activating antigen binding
construct described herein. The label or package insert indicates
that the composition is used for treating the condition of choice.
Moreover, the article of manufacture may comprise (a) a first
container with a composition contained therein, wherein the
composition comprises an antigen-binding construct described
herein; and (b) a second container with a composition contained
therein, wherein the composition comprises a further cytotoxic or
otherwise therapeutic agent. The article of manufacture in this
embodiment described herein may further comprise a package insert
indicating that the compositions can be used to treat a particular
condition. Alternatively, or additionally, the article of
manufacture may further comprise a second (or third) container
comprising a pharmaceutically-acceptable buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
Use of the TDP-43-Binding Constructs in the Diagnosis and
Monitoring of TDP-43 Proteinopathies
[0205] TDP-43 antigen-binding constructs may be used in the
diagnosis of TDP-43 proteinopathies, particularly amyotrophic
lateral sclerosis and/or frontotemporal lobar degeneration.
Provided herein is the use of the TDP-43 antigen-binding constructs
in the diagnosis and/or monitoring of a subject predisposed or
suspected of developing a neurodegenerative disease or suffering
from a neurodegenerative disease. The TDP-43 antigen-binding
constructs may also be used in monitoring the efficacy of a
treatment administered to a subject suffering from a TDP
proteinopathy.
[0206] Assay methods are provided in co-owned patent application
published as WO 2012/174666A1 (herein incorporated by reference)
for determining the level of interaction between a TDP-43
polypeptide or fragment thereof and a NF-kB p65 polypeptide or
fragment thereof in a biological sample of the subject. Such assay
methods may employ a TDP-43 binding construct described herein. An
elevated level of interaction between TDP-43 polypeptide or
fragment thereof and p65 polypeptide or fragment thereof in the
biological sample relative to a reference level of interaction
between TDP-43 polypeptide or fragment thereof and p65 polypeptide
or fragment thereof indicates that the subject is predisposed or
suspected of developing a neurodegenerative disease or is suffering
from a neurodegenerative disease. Further methods of using the
TDP-43 antigen-binding constructs described herein to detect the
association of TDP-43 and NF-kB are provided in the Examples
below.
[0207] In another embodiment the TDP-43 antigen-binding constructs
or nucleic acids encoding them may be used in a diagnostic
composition as reagents in immuno- or nucleic acid-based diagnostic
methods. The TDP-43 antigen-binding constructs as described herein
are, for example, suited for use in immunoassays in which they can
be utilized in liquid phase or bound to a solid phase carrier.
Examples of immunoassays which can utilize the TDP-43
antigen-binding constructs as described herein are competitive and
non-competitive immunoassays in either a direct or indirect format.
Examples of such immunoassays are the radioimmunoassay (RIA), the
sandwich (immunometric assay), flow cytometry and the Western blot
assay.
[0208] The antigen-binding constructs as described herein may be
labeled for use in an assay. There are many different labels and
methods of labeling known to those of ordinary skill in the art.
Examples of the types of labels which can be used inone embodiment
include enzymes, radioisotopes, colloidal metals, fluorescent
compounds, chemiluminescent compounds, and bioluminescent
compounds.
[0209] In a further embodiment, the TDP-43 antigen-binding
constructs as described herein can also be used in a method for the
diagnosis of a disorder in an individual by obtaining a sample from
the tested individual which can be a blood sample, a lymph sample,
cerebrospinal fluid, or a neural tissue biopsy sample and
contacting the sample with a TDP-43 antigen-binding constructs
under conditions enabling the formation of antibody-antigen
complexes. The level of such complexes is then determined by
methods known in the art. A level significantly higher than that
formed in a control sample indicating the disease in the tested
individual. One embodiment, is a method of diagnosing a TDP-43
proteinopathy in a subject, the method comprising: (a) assessing
the level of TDP-43 in a sample from the subject to be diagnosed
with an TDP-43 antigen-binding construct, and (b) comparing the
level of TDP-43 observed to a reference standard that indicates the
level of the TDP-43 in one or more control subjects, wherein a
difference or similarity between the level of the TDP-43 and the
reference standard indicates that the subject suffers from a TDP-43
proteinopathy. The subject to be diagnosed can be asymptomatic or
preclinical for the disease. In one embodiment, the control subject
has a TDP-43 proteinopathy, for example ALS or FTLD, wherein a
similarity between the level of TDP-43 and the reference standard
indicates that the subject, to be diagnosed has a TDP-43
proteinopathy.
[0210] The level of TDP-43 can be assessed by any suitable method
known in the art comprising, e.g., analyzing TDP-43 by one or more
techniques chosen from Western blot, immunoprecipitation,
enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
fluorescent activated cell sorting (FACS), or gel
electrophoresis.
EXAMPLES
[0211] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for. The practice of the present invention will employ,
unless otherwise indicated, conventional methods of protein
chemistry, biochemistry, recombinant DNA techniques and
pharmacology, within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., T. E. Creighton,
Proteins: Structures and Molecular Properties (W.H. Freeman and
Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,
Inc., current addition); Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.
Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's
Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing
Company, 1990); Carey and Sundberg Advanced Organic Chemistry
3.sup.rd Ed. (Plenum Press) Vols A and B(1992).
Example 1: Generation of Exemplary Monoclonal Antibodies Against
the TDP-43 RRM1 Domain of TDP-43
[0212] A series of exemplary monoclonal antibodies against a
recombinant protein encoding the RRMI domain of TDP-43 were
generated. The TDP-43 fragment used as antigen was generated and
purified as follows. The EcoR1-Not1 428 bp hTDP43 cDNA fragment
encoding RRM1 was PCR-amplified and cloned into the pGEX-6P-1
vector (GE Healthcare). This recombinant plasmid was used to
transform the E. coli host BL21 for the expression of the 136 aa
protein encoding all the RRM1 domain of hTDP43. The 15.68 kDa
recombinant protein was subsequently purified using the Glutathione
S-transferase (GST) gene fusion system (GE Healthcare).
[0213] C57B16 mice were immunized with this polypeptide fragment,
comprising amino acids 40-176 of TDP-43 (see Table A for sequence).
The exemplary anti-TDP-43 monoclonal antibodies that were obtained
using standard hybridoma technology yielded an immunodetection
signal for TDP-43 protein when tested by immunoblotting after
SDS-PAGE of spinal cord extracts from non-transgenic mice, as well
as from transgenic mice over-expressing human TDP-43 (data not
shown). Antibodies from three hybridoma clones C10, G8 and E6 also
detected TDP-43 on immunoblots of nuclear extracts from mouse
microglial BV-2 cells fractionated by SDS-PAGE (shown in FIG.
1).
Example 2: Human Recombinant NF-.kappa.B p65 Interacts Directly
with Human Recombinant TDP-43
[0214] BSA 0.8 ug/ml or human recombinant TDP-43 in serial
dilutions from 0.8 ug/ml to 0.0125 ug/ml were prepared in PBS,
loaded on ELISA microplates (6 wells per conditions) and incubated
overnight. Human recombinant p65-His-Tag (Enzo Life Sciences) was
prepared at the concentration of 0.2 ug/ml, added to all wells and
incubated 2h. After 2h incubation in Blocking Buffer,
anti-His-Tag-HRP (1:10.000) (Abcam) was prepared in Diluent
Solutions, loaded on the plate and incubated 2h. Substrate Solution
was added and Absorbance read at 450 nm. The results are shown in
FIG. 2. Values are expressed as mean.+-.sem.
Example 3: Interaction Between Recombinant TDP-43 and NF-.kappa.B
p65 is Inhibited by Exemplary Bivalent Monoclonal Anti-RRM1-TDP-43
Antibodies C10, G8 and E6
[0215] Human recombinant TDP-43 was prepared at the concentration
of 0.2 ug/ml, loaded on ELISA microplates and incubated overnight.
p65-His-Tag (0.4 ug/ml) was mixed 1:1 with PBS, BSA (0.4 ug/ml),
anti-TDP-43 polyclonal antibodies (Proteintech) (0.4 ug/ml IgG) or
anti-RRM1-TDP-43 monoclonal antibodies (C10, G8 or E6 monoclonals
having an Fab/Fab format with an Fc domain) (0.4 ug/ml IgG) to
reach the final concentration of 0.2 ug/ml p65 and 0.2 ug/ml
interfering antibody. Mixed solutions were loaded on the plate (8
wells per condition) and incubated 2h. After 2h incubation in
Blocking Buffer, anti-His-Tag-HRP (1:10.000) is prepared in Diluent
Solutions, loaded on the plate and incubated 2h. Substrate Solution
is added and Absorbance read at 450 nm. The results are shown in
FIG. 3, and demonstrate that all 3 of the monoclonal antibodies
C10, G8 and E6 block the interaction between TDP-43 and NF-.kappa.B
p65 to a much greater extent than the polyclonal anti-TDP antibody.
Values are expressed as mean.+-.sem.
Example 4: Derivation of Single Chain Anti-TDP-43 Antibodies
[0216] The mRNAs were isolated from the appropriate hybridoma cell
lines from Example 1 to derive cDNAs encoding scFv antibodies under
the control of a CMV promoter. The variable regions of heavy chain
(VH) and light chain (Vk) were amplified separately from
first-strand cDNA by using a mixture of PCR primers and were cloned
into the pBZUT7 (as described in Patel et al. Mol Ther., 22 (3),
498-510 2014) vector and sequenced. The VH and Vk domains were
assembled and linked together to yield a full-length scFv gene. The
scFv gene was constructed in a VH-linker-Vk format together with a
standard flexible 20-amino acid linker (Gly4Ser)3 and it was then
subcloned upstream of the Myc-tagged Psw1 scFVD1.3 Tag1 expression
vector (Patel et al. op. cit.) to generate scFv-TDP-43. Exemplary
clones were then sequenced. The scFv generated contained a murine
immunoglobulin (Ig) .kappa.-secretory signal for efficient
secretion and a human c-myc epitope to facilitate detection. A
schematic drawing of these constructs is shown in FIG. 4.
Example 5: Anti-TDP-43 Antibodies Having an scFv Format Localize in
the Cytoplasm and Nucleus of HEK 293 Cells, and are Secreted
[0217] Hek 293 cells were transiently transfected with pScFv9
expression plasmid containing Ig.kappa. domain, Myc-tag and a
combination of the two VH and V.kappa. obtained from the E6 clone,
i.e. VH1Vk9, VH1Vk11, VH7Vk9 and VH7Vk11. A schematic
representation of the constructs is shown in FIG. 4A. Cells were
transfected with 4 .mu.g of plasmid for 48h and then collected.
Cytoplasmic and nuclear fractions were obtained and loaded on a 12%
gel. Rabbit polyclonal anti-myc antibody (Abcam) was used to detect
the ScFv antibodies inside the cells. Media of transfected cells
were collected, centrifuged at 5000 rpm for 15 min at 4.degree. C.
to eliminate debris and precipitated over-night at -20.degree. C.
The pellet was resuspended in loading buffer and loaded on 12% gel.
The scFv antibodies were detected by blotting the membrane with
mouse monoclonal anti-myc antibody (Santa Cruz). The results show
that E6_VH1V.kappa.9 and E6_VH7V.kappa.9 scFv antibodies localized
in both the cytoplasm and the nucleus of Hek 293 cells (FIG. 5).
The scFv antibodies were also secreted into the medium (FIG.
5).
Example 6: Anti-TDP-43 Antibodies Having an scFv Format Detect
TDP-43 RRM1 Domain
[0218] Different concentrations of TDP-43 1-206 amino acid
fragment, containing the RRM1 domain were loaded onto membranes by
dot blot. Membranes were incubated overnight with media from Hek293
cells that had been transfected with pScFv9 expression plasmid
containing the nucleic acids encoding E6_VH1Vk9 and E6_VH7Vk9. The
myc signal was detected using anti-myc HRP antibody incubation. The
results, shown in FIG. 6, demonstrate that E6_VH1Vk9 and E6_VH7Vk9
scFv antibodies are able to recognize specifically TDP-43.
Example 7: E6_VH1Vk9 and E6_VH7Vk9 Exemplary Antigen-Binding
Constructs in an scFv Format Block the Interaction of TDP-43 with
NF-.kappa.B p65
[0219] An ELISA assay was performed as described in EXAMPLE 2 using
the conditioned medium from E6_VH1Vk9- and E6_VH7Vk9-expressing
cells as a source of the antibodies. Medium from HEK293 cells that
had been transfected with an empty pScFv9 cells, or an irrelevant
insert (D1.3) were used as controls. The results show that both
E6_VH1Vk9 and E6_VH7Vk9 are capable of blocking the TDP-43
interaction with NF-.kappa.B p65 (FIG. 7). The bivalent Fab/Fab
format antibody E6 at a concentration of 0.4 ug/ml inhibited the
TDP-43 interaction with NF-.kappa.B p65 to an even greater extent,
and a TDP-43 polyclonal antibody inhibited, but to a lesser degree
than the monoclonals.
Example 8: Antibodies Expressed in HEK293 Cells Interact with
TDP-43 Intracellularly
[0220] Cell lysates were made from E6_VH1Vk9- and
E6_VH7Vk9-expressing HEK293 cells. The lysates were exposed to
polyclonal anti-TDP 43 to immunoprecipate cellular TDP-43.
Immunopreciptates were resolved using PAGE. The results are shown
in FIG. 8, demonstrating that the scFv antibodies E6_VH1Vk9 and
E6_VH7Vk9 (detected by a myc tag) were co-immunoprecipitated with
TDP-43. This demonstrates that the antigen-binding constructs
E6_VH1V.kappa.9 and E6_VH7V.kappa.9 bound to intracellular
TDP-43.
Example 9: VH1Vk9 Antibody Blocks the Interaction of TDP-43 with
NF-.kappa.B p65 in HEK293 Cells
[0221] Cell lysates were made from E6_VH1Vk9-expressing HEK293
cells which have been treated with TNF.alpha. 4 hours. The lysates
were exposed to polyclonal anti-TDP 43 antibody to immunoprecipate
cellular TDP-43. Immunoprecipitates were resolved using PAGE. FIG.
9 shows an immunoblot in which an antibody against p65 was used to
detect immunoprecipitates. The results show that decreased levels
of NF-.kappa.B p65 were present when VH1Vk9 was expressed in the
HEK293 cells. This indicates that VH1Vk9 interfered with the
binding of TPD-43 to NF-.kappa.B p65 in the cell.
Example 10: Inhibition of NF-.kappa.B Activation by scFv Exemplary
Antibodies Against TDP-43 in Cultured Cell Systems
[0222] A cell line was previously generated by stable transfection
of BV-2 microglial cells with stable insertion of a luciferase
reporter 4 kBwt luciferase plasmid and subsequent selection with
hygomycin (Swamp, 2011 J. Exp. Med. op cit.). Expression plasmid
vectors encoding scFv anti-TDP-43 antibodies were transfected into
these BV-2 cultured cells and transiently expressed for 48 hours.
During the final 4 hours, cells were exposed to either PBS
(control) or 500 ng/ml LPS. Cells were lysed with Glo Buffer
(Promega) and 50 .mu.l of lysates were loaded in replicates on a 96
well plate. Luciferase substrate was added following the assay
procedures (Bright-Glo Luciferase Assay, Promega). RLU (relative
light units) of luminescence were determined using an automatic
plate reader and normalized on total proteins (.mu.g) present in
the well, as determined by protein quantification (Biorad).
[0223] The results shown in FIG. 10 demonstrate the activation of
the NF-.kappa.B reporter gene was reduced after treatment with LPS
in BV2 cells expressing either scFv antibody VH1V.kappa.9 or
E6_VH7V.kappa.9. There was no effect of the antibodies on viability
of BV2 cells (data not shown). We note that the scFv VH7-Vk9
antibody was more effective in reducing NF-.kappa.B activity of BV2
cells than the scFv VH1Vk9 antibody. The scFv VH7-Vk9 attenuated
NF-.kappa.B activity by 32% whereas scFv VH1Vk9 reduced NF-.kappa.B
activity by 13%. This is intriguing because these two scFv
antibodies differ by only one amino acid, a Q (glutamine) instead
of E (glutamic acid) in the scFv VH7-Vk9. Somehow this minor
sequence variation appears to enhance the propensity of scFv
E6_VH7Vk9 to distribute in the nucleus (as shown in FIG. 5), a
factor that may explain a more efficient inhibition of TDP-43
interaction with NF-.kappa.B p65 in the nucleus.
Example 11: Expression of scFv Antibodies in Neuro2A Cells Caused
Reduction in Levels of Nuclear TDP-43
[0224] Neuro 2A cells were transiently trasfected with E6_VH1Vk9,
E6_Vh7Vk9 or an empty vector as in Example 5. After 48 hours,
nuclear extracts were obtained from the cells and the amount of
nuclear TDP-43 was quantified by ponceau staining. The results are
shown in FIG. 11. Both E6_VH1Vk9, E6_Vh7Vk9 reduced the amount of
nuclear TDP-43 significantly. In ALS, there is an upregulation of
TDP-43 mRNA and protein levels (Swarup et al. J Exp med 2011). This
result suggests that anti-TDP-43 scFv antibodies might confer
protection by attenuating the upregulation of TDP-43 in ALS.
Example 12: Production of Adeno-Associated Viral AAV Vectors
Containing scFv Antibodies
[0225] The following protocol was used to produce pscAAV vectors
containing E6-derived single chain antibodies. First, to produce
unsecreted single chain antibodies (-IgK), a 773 bp fragment was
obtained from pScFv9_E6VH1Vk9 and pScFv9_E6VHVk9 by PCR introducing
XbaI restriction site before the VH sequence and NotI at the end of
the myc sequence. Digestion of the scAAV-CMV-EGFP (described in
McCarty et al. Gene Ther. 8: 1248-1254, 2001) with XbaI and NotI
restriction enzymes allowed replacing the EGFP sequence with
E6VH1Vk9(-IgK) and E6VH7Vk9(-IgK). Subsequently, the IgK sequence
was obtained by pscFv9 plasmid using HindIII and PstI restriction
enzymes. A XbaI/PstI fragment containing HindIII restriction site
was inserted into corresponding restriction sites of
pscAAV_E6VH1Vk9_NS and E6VH7Vk9 NS plasmids before the VH sequence.
Finally, pscAAV plasmids were digested with HindIII and PstI and
the IgK sequence was inserted to obtain pscAAV_E6VH1Vk9 and
E6VH7Vk9 with a secretion signal. The secreted single chain
antibodies were obtained by a standard method (Rabinowitz et al. J
Virol., 76: 791-801(2002)). Briefly, the fragment encompassing the
ScFv expression cassette in pscFv9-E6VH1Vk9 and E6VH7Vk9 was
excised using HindIII and EcoRV and cloned into the plasmid
Bluescript SK(-) (Stratagene, Canada). It was then recloned as a
SalI/NotI fragment into the XhoI/NotI digested scAAV-CMV-EGFP
plasmid (McCarty et al. Gene Ther., 2011) replacing the EGFP
encoding sequence and creating the pscAAV_E6VH1Vk9 and
pscAAV_E6VH7Vk9 plasmids to be used in production of AAV
recombinant viruses. Sequencing confirmed the correct insertions
inside the plasmids.
Example 13: E6 scFv Antibodies Reduce TDP-43 Insolubility in
Ethacrynic Acid-Treated Cells
[0226] Hek293 cells were transfected 72h in Optimem with 2 .mu.g
DNA and 5 .mu.l Lipofectamine 2000 reagent (Invitrogen) with E6_V1
and E6_V7 scFv antibodies, empty plasmid or anti D1.3 scFv antibody
and treated overnight with 50 .mu.M ethacrynic acid (EA) to induce
TDP-43 aggregation, or treated overnight with same volume of DMSO
diluent as a control. The RIPA (radio-immunoprecipitation assay
buffer) insoluble fraction was obtained. A representative western
blot is shown in FIG. 12A and a representative dot blot analysis of
TDP-43 is shown in FIG. 12B. The results show that both the E6_V1
and E6_V7 scFv antibodies reduced the amount of insoluble TDP-43
detected after EA treatment (n=3/conditions). *, p<0.05 versus
Empty.
[0227] Immunofluorescence staining of the Hek293 transfected cells
was conducted to localize TDP-43 in the cells (data not shown). In
untransfected cells, TDP-43 mis-localized in the cytoplasm after EA
treatment. However, in transfected cells expressing E6 V1 and E6_V7
TDP-43 remained in the nucleus after EA treatment, avoiding
cytoplasmic localization and aggregation.
Example 14: E6 ScFv Antibodies Protect TDP-43 from Lysine
Acetylation
[0228] Hek293 cells were transfected 48h in Optimem with 2 .mu.g
DNA and 5 .mu.l Lipofectamine 2000 reagent (Invitrogen) with E6
scFv antibodies V1 and V7, empty plasmid or anti D1.3 scFv antibody
and treated for 4 hours with 50 ng/ml TNF alpha. Lysine acetylated
proteins were immunoprecipitated using anti-acetyl-lysine antibody
(Millipore) overnight in 300 .mu.g total proteins lysate and the
presence of lysine-acetylated TDP-43 was detected by western
blotting for TDP-43 (Figure. 13). This result shows that that the
E6 scFv antibodies protected TDP-43 from lysine acetylation. An
empty transfection vector (EMT) was not protective, nor was a
transfected control antibody D1.3. Lysine acetylation has been
proposed as a novel post-translational modification controlling
TDP-43 function and aggregation, and there is evidence that TDP-43
acetylation impairs RNA-binding and promotes accumulation of
insoluble, hyper-phosphorylated TDP-43 species that largely
resemble pathological inclusions in ALS and FTLD-TDP (Cohen T. J.,
Hwang A. W., Restrepo C. R., et al. Nat Com 2014).
[0229] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent
application publications, U.S. patent application, foreign patents,
foreign patent application and non-patent publications referred to
in this specification and/or listed in the Application Data
Sheetare incorporated herein by reference, in their entirety.
Aspects of the embodiments can be modified, if necessary to employ
concepts of the various patents, application and publications to
provide yet further embodiments. In the case of inconsistencies,
the present disclosure will prevail.
[0230] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. The scope of the claims should
not be limited by the preferred embodiments set forth in the
examples, but should be given the broadest interpretation
consistent with the description as a whole.
* * * * *